ROS-induced PADI2 downregulation accelerates cellular senescence via the stimulation of SASP production and NFκB activation.

Cellular senescence is closely related to tissue aging including bone. Bone homeostasis is maintained by the tight balance between bone-forming osteoblasts and bone-resorbing osteoclasts, but it undergoes deregulation with age, causing age-associated osteoporosis, a main cause of which is osteoblast dysfunction. Oxidative stress caused by the accumulation of reactive oxygen species (ROS) in bone tissues with aging can accelerate osteoblast senescence and dysfunction. However, the regulatory mechanism that controls the ROS-induced senescence of osteoblasts is poorly understood. Here, we identified Peptidyl arginine deiminase 2 (PADI2), a post-translational modifying enzyme, as a regulator of ROS-accelerated senescence of osteoblasts via RNA-sequencing and further functional validations. PADI2 downregulation by treatment with H2O2 or its siRNA promoted cellular senescence and suppressed osteoblast differentiation. CCL2, 5, and 7 known as the elements of the senescence-associated secretory phenotype (SASP) which is a secretome including proinflammatory cytokines and chemokines emitted by senescent cells and a representative feature of senescence, were upregulated by H2O2 treatment or Padi2 knockdown. Furthermore, blocking these SASP factors with neutralizing antibodies or siRNAs alleviated the senescence and dysfunction of osteoblasts induced by H2O2 treatment or Padi2 knockdown. The elevated production of these SASP factors was mediated by the activation of NFκB signaling pathway. The inhibition of NFκB using the pharmacological inhibitor or siRNA effectively relieved H2O2 treatment- or Padi2 knockdown-induced senescence and osteoblast dysfunction. Together, our study for the first time uncover the role of PADI2 in ROS-accelerated cellular senescence of osteoblasts and provide new mechanistic and therapeutic insights into excessive ROS-promoted cellular senescence and aging-related bone diseases.

CC-chemokine class inhibition attenuates pathological angiogenesis while preserving physiological angiogenesis.

Increasing evidence shows that CC-chemokines promote inflammatory-driven angiogenesis, with little to no effect on hypoxia-mediated angiogenesis. Inhibition of the CC-chemokine class may therefore affect angiogenesis differently depending on the pathophysiological context. We compared the effect of CC-chemokine inhibition in inflammatory and physiological conditions. In vitro, the broad-spectrum CC-chemokine inhibitor "35K" inhibited inflammatory-induced endothelial cell proliferation, migration, and tubulogenesis, with more modest effects in hypoxia. In vivo, adenoviruses were used to overexpress 35K (Ad35K) and GFP (AdGFP, control virus). Plasma chemokine activity was suppressed by Ad35K in both models. In the periarterial femoral cuff model of inflammatory-driven angiogenesis, overexpression of 35K inhibited adventitial neovessel formation compared with control AdGFP-infused mice. In contrast, 35K preserved neovascularization in the hindlimb ischemia model and had no effect on physiological neovascularization in the chick chorioallantoic membrane assay. Mechanistically, 2 key angiogenic proteins (VEGF and hypoxia-inducible factor-1α) were conditionally regulated by 35K, such that expression was inhibited in inflammation but was unchanged in hypoxia. In conclusion, CC-chemokine inhibition by 35K suppresses inflammatory-driven angiogenesis while preserving physiological ischemia-mediated angiogenesis via conditional regulation of VEGF and hypoxia-inducible factor-1α. CC-chemokine inhibition may be an alternative therapeutic strategy for suppressing diseases associated with inflammatory angiogenesis without inducing the side effects caused by global inhibition.- Ridiandries, A., Tan, J. T. M., Ravindran, D., Williams, H., Medbury, H. J., Lindsay, L., Hawkins, C., Prosser, H. C. G., Bursill, C. A. CC-chemokine class inhibition attenuates pathological angiogenesis while preserving physiological angiogenesis.

CCR3 is a target for age-related macular degeneration diagnosis and therapy.

Age-related macular degeneration (AMD), a leading cause of blindness worldwide, is as prevalent as cancer in industrialized nations. Most blindness in AMD results from invasion of the retina by choroidal neovascularisation (CNV). Here we show that the eosinophil/mast cell chemokine receptor CCR3 is specifically expressed in choroidal neovascular endothelial cells in humans with AMD, and that despite the expression of its ligands eotaxin-1, -2 and -3, neither eosinophils nor mast cells are present in human CNV. Genetic or pharmacological targeting of CCR3 or eotaxins inhibited injury-induced CNV in mice. CNV suppression by CCR3 blockade was due to direct inhibition of endothelial cell proliferation, and was uncoupled from inflammation because it occurred in mice lacking eosinophils or mast cells, and was independent of macrophage and neutrophil recruitment. CCR3 blockade was more effective at reducing CNV than vascular endothelial growth factor A (VEGF-A) neutralization, which is in clinical use at present, and, unlike VEGF-A blockade, is not toxic to the mouse retina. In vivo imaging with CCR3-targeting quantum dots located spontaneous CNV invisible to standard fluorescein angiography in mice before retinal invasion. CCR3 targeting might reduce vision loss due to AMD through early detection and therapeutic angioinhibition.

Inhibition of Epithelial CC-Family Chemokine Synthesis by the Synthetic Chalcone DMPF-1 via Disruption of NF-κB Nuclear Translocation and Suppression of Experimental Asthma in Mice.

Asthma is associated with increased pulmonary inflammation and airway hyperresponsiveness. The interaction between airway epithelium and inflammatory mediators plays a key role in the pathogenesis of asthma. In vitro studies evaluated the inhibitory effects of 3-(2,5-dimethoxyphenyl)-1-(5-methylfuran-2-yl)prop-2-en-1-one (DMPF-1), a synthetic chalcone analogue, upon inflammation in the A549 lung epithelial cell line. DMPF-1 selectively inhibited TNF-α-stimulated CC chemokine secretion (RANTES, eotaxin-1, and MCP-1) without any effect upon CXC chemokine (GRO-α and IL-8) secretion. Western blot analysis further demonstrated that the inhibitory activity resulted from disruption of p65NF-κB nuclear translocation without any effects on the mitogen-activated protein kinase (MAPK) pathway. Treatment of ovalbumin-sensitized and ovalbumin-challenged BALB/c mice with DMPF-1 (0.2-100 mg/kg) demonstrated significant reduction in the secretion and gene expression of CC chemokines (RANTES, eotaxin-1, and MCP-1) and Th2 cytokines (IL-4, IL-5, and IL-13). Furthermore, DMPF-1 treatment inhibited eosinophilia, goblet cell hyperplasia, peripheral blood total IgE, and airway hyperresponsiveness in ovalbumin-sensitized and ovalbumin-challenged mice. In conclusion, these findings demonstrate the potential of DMPF-1, a nonsteroidal compound, as an antiasthmatic agent for further pharmacological evaluation.

Interleukin-17 is a negative regulator of established allergic asthma.

T helper (Th)17 cells producing interleukin (IL)-17 play a role in autoimmune and allergic inflammation. Here, we show that IL-23 induces IL-17 in the lung and IL-17 is required during antigen sensitization to develop allergic asthma, as shown in IL-17R-deficient mice. Since IL-17 expression increased further upon antigen challenge, we addressed its function in the effector phase. Most strikingly, neutralization of IL-17 augmented the allergic response in sensitized mice. Conversely, exogenous IL-17 reduced pulmonary eosinophil recruitment and bronchial hyperreactivity, demonstrating a novel regulatory role of IL-17. Mechanistically, IL-17 down modulated eosinophil-chemokine eotaxin (CCL11) and thymus- and activation-regulated chemokine/CCL17 (TARC) in lungs in vivo and ex vivo upon antigen restimulation. In vitro, IL-17 reduced TARC production in dendritic cells (DCs)-the major source of TARC-and antigen uptake by DCs and IL-5 and IL-13 production in regional lymph nodes. Furthermore, IL-17 is regulated in an IL-4-dependent manner since mice deficient for IL-4Ralpha signaling showed a marked increase in IL-17 concentration with inhibited eosinophil recruitment. Therefore, endogenous IL-17 is controlled by IL-4 and has a dual role. Although it is essential during antigen sensitization to establish allergic asthma, in sensitized mice IL-17 attenuates the allergic response by inhibiting DCs and chemokine synthesis.

Pan-CC chemokine neutralization restricts splenocyte egress and reduces inflammation in a model of arthritis.

Chemokines are key regulators of leukocyte trafficking and play a crucial role under homeostatic and inflammatory conditions. Because chemokines are involved in multiple pathologies, they represent an attractive class of therapeutic targets. However, because of the redundancy of this system, neutralizing a single chemokine may be insufficient to achieve therapeutic benefit. Our strategy was to use a Fc-fusion recombinant protein form of the poxvirus-derived viral CC chemokine inhibitor protein (vCCI-Fc) that has the ability to specifically bind to multiple CC chemokines and neutralize their activity. In this study, we demonstrate first that, in vivo, vCCI-Fc prevents CC chemokine-dependent migration of macrophages into inflamed tissue of carageenan-challenged mice. We next studied this effect of inhibiting CC chemokine activity in a model more relevant to human disease, collagen-induced arthritis. Mice receiving vCCI-Fc revealed a striking retention of splenocytes, including activated and IFN-gamma-secreting CD4(+) and CD8(+) T cells, that was associated with a concomitant decrease of cells in the draining lymph nodes. These phenomena resulted in a significant decrease in the incidence of disease and a reduction in clinical score, joint inflammation, and cartilage destruction as compared with mice receiving isotype control. Taken together, these results define a role for CC chemokines in the control of disease, as interfering with their function leads to a previously unappreciated role of controlling inflammatory cell trafficking in and out of secondary lymphoid organs.

In silico design of multi-target inhibitors for C-C chemokine receptors using substructural descriptors.

Rational design of entry inhibitors is an active area for the discovery of new and effective anti-HIV agents. C-C Chemokine receptors represent key targets for the HIV entry process. Several of these proteins with features to be HIV co-receptors have not been sufficiently studied or used for the design of novel entry inhibitors. With the purpose to overcome this problem, we develop here a fragment-based approach for the design of multi-target inhibitors against four C-C chemokine receptors. This approach was focused on the construction of a multi-target QSAR discriminant model using a large and heterogeneous database of compounds and substructural descriptors for the classification and prediction of inhibitors for C-C chemokine receptors. The model correctly classified more than 89% of active and inactive compounds in both: training and prediction series. As principal advantage, this model permitted the automatic and fast extraction of fragments responsible for the inhibitory activity against the different C-C chemokine receptors under study and new molecular entities were suggested as possible versatile inhibitors for these proteins.

Site-directed mutagenesis of the CC chemokine binding protein 35K-Fc reveals residues essential for activity and mutations that increase the potency of CC chemokine blockade.

Chemokines of the CC class are key mediators of monocyte recruitment and macrophage differentiation and have a well documented role in many inflammatory diseases. Blockade of chemokine activity is therefore an attractive target for anti-inflammatory therapy. 35K (vCCI) is a high-affinity chemokine binding protein expressed by poxviruses, which binds all human and murine CC chemokines, preventing their interaction with chemokine receptors. We developed an Fc-fusion protein of 35K with a modified human IgG1 Fc domain and expressed this construct in human embryonic kidney 293T cells. Purified 35K-Fc is capable of inhibiting CC chemokine-induced calcium flux, chemotaxis, and ß-arrestin recruitment in primary macrophages and transfected cells. To elucidate the residues involved in chemokine neutralization, we performed site-directed mutagenesis of six key amino acids in 35K and expressed the mutant Fc-fusion proteins in vitro. We screened the mutants for their ability to block chemokine-induced ß-arrestin recruitment in transfected cells and to inhibit primary macrophage signaling in an electric cell substrate impedance sensing assay. Using a sterile model of acute inflammation, zymosan-induced peritonitis, we confirmed that wild-type 35K-Fc can reduce monocyte recruitment, whereas one mutant (R89A) showed a more pronounced blockade of monocyte influx and another mutant (E143K) showed total loss of function. We believe that 35K-Fc will be a useful tool for exploring the role of CC chemokines in chronic inflammatory pathologies, and we have identified a higher potency form of the molecule that may have potential therapeutic applications in chronic inflammatory disease.

IκBα glutathionylation and reduced histone H3 phosphorylation inhibit eotaxin and RANTES.

Airway smooth muscle cells (ASMCs) secrete eotaxin and RANTES (regulated on activation, normal T-cell expressed and secreted) in response to tumour necrosis factor (TNF)-α, which is inhibited by the nuclear factor (NF)-κB inhibitor dimethylfumarate (DMF). NF-κB/IκB (inhibitor of NF-κB) glutathionylation and changes in chromatin remodelling can inhibit NF-κB activity. In this study, we determined whether NF-κB/IκB glutathionylation and reduced histone H3 phosphorylation might underlie the inhibitory effect of DMF on NF-κB activity, and eotaxin and RANTES secretion. Primary human ASMCs were treated with DMF, diamide and/or glutathione (GSH) ethylester (OEt) prior to TNF-α stimulation and were subsequently analysed by ELISA, electrophoretic mobility shift assay, immunofluorescence, co-immunoprecipitation or immunoblotting. DMF reduced intracellular GSH and induced IκBα glutathionylation (IκBα-SSG), which inhibited IκBα degradation, NF-κB p65 nuclear entry and NF-κB/DNA binding. In addition, DMF inhibited the phosphorylation of histone H3, which was possibly mediated by the inhibitory effect of DMF on mitogen- and stress-activated protein kinase (MSK)-1. However, p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase MAPK and MAPK phosphatase-1, upstream of MSK-1, were not inhibited by DMF. Importantly, DMF-mediated effects on NF-κB, histone H3, eotaxin and RANTES were reversed by addition of GSH-OEt. Our data suggest that DMF inhibits NF-κB-dependent eotaxin and RANTES secretion by reduction of GSH with subsequent induction of IκBα-SSG and inhibition of histone H3 phosphorylation. Our findings offer new potential drug targets to reduce airway inflammation in asthma.

The coordinated action of CC chemokines in the lung orchestrates allergic inflammation and airway hyperresponsiveness.

The complex pathophysiology of lung allergic inflammation and bronchial hyperresponsiveness (BHR) that characterize asthma is achieved by the regulated accumulation and activation of different leukocyte subsets in the lung. The development and maintenance of these processes correlate with the coordinated production of chemokines. Here, we have assessed the role that different chemokines play in lung allergic inflammation and BHR by blocking their activities in vivo. Our results show that blockage of each one of these chemokines reduces both lung leukocyte infiltration and BHR in a substantially different way. Thus, eotaxin neutralization reduces specifically BHR and lung eosinophilia transiently after each antigen exposure. Monocyte chemoattractant protein (MCP)-5 neutralization abolishes BHR not by affecting the accumulation of inflammatory leukocytes in the airways, but rather by altering the trafficking of the eosinophils and other leukocytes through the lung interstitium. Neutralization of RANTES (regulated upon activation, normal T cell expressed and secreted) receptor(s) with a receptor antagonist decreases significantly lymphocyte and eosinophil infiltration as well as mRNA expression of eotaxin and RANTES. In contrast, neutralization of one of the ligands for RANTES receptors, macrophage-inflammatory protein 1alpha, reduces only slightly lung eosinophilia and BHR. Finally, MCP-1 neutralization diminishes drastically BHR and inflammation, and this correlates with a pronounced decrease in monocyte- and lymphocyte-derived inflammatory mediators. These results suggest that different chemokines activate different cellular and molecular pathways that in a coordinated fashion contribute to the complex pathophysiology of asthma, and that their individual blockage results in intervention at different levels of these processes.

Interaction of chemokine receptor CCR5 with its ligands: multiple domains for HIV-1 gp120 binding and a single domain for chemokine binding.

CCR5 is a chemokine receptor expressed by T cells and macrophages, which also functions as the principal coreceptor for macrophage (M)-tropic strains of HIV-1. To understand the molecular basis of the binding of chemokines and HIV-1 to CCR5, we developed a number of mAbs that inhibit the various interactions of CCR5, and mapped the binding sites of these mAbs using a panel of CCR5/CCR2b chimeras. One mAb termed 2D7 completely blocked the binding and chemotaxis of the three natural chemokine ligands of CCR5, RANTES (regulated on activation normal T cell expressed and secreted), macrophage inflammatory protein (MIP)-1alpha, and MIP-1beta, to CCR5 transfectants. This mAb was a genuine antagonist of CCR5, since it failed to stimulate an increase in intracellular calcium concentration in the CCR5 transfectants, but blocked calcium responses elicited by RANTES, MIP-1alpha, or MIP-1beta. This mAb inhibited most of the RANTES and MIP-1alpha chemotactic responses of activated T cells, but not of monocytes, suggesting differential usage of chemokine receptors by these two cell types. The 2D7 binding site mapped to the second extracellular loop of CCR5, whereas a group of mAbs that failed to block chemokine binding all mapped to the NH2-terminal region of CCR5. Efficient inhibition of an M-tropic HIV-1-derived envelope glycoprotein gp120 binding to CCR5 could be achieved with mAbs recognizing either the second extracellular loop or the NH2-terminal region, although the former showed superior inhibition. Additionally, 2D7 efficiently blocked the infectivity of several M-tropic and dual-tropic HIV-1 strains in vitro. These results suggest a complicated pattern of HIV-1 gp120 binding to different regions of CCR5, but a relatively simple pattern for chemokine binding. We conclude that the second extracellular loop of CCR5 is an ideal target site for the development of inhibitors of either chemokine or HIV-1 binding to CCR5.

Intratumoral delivery of CCL25 enhances immunotherapy against triple-negative breast cancer by recruiting CCR9+ T cells.

CCR9+ T cells have an increased potential to be activated and therefore may mediate strong antitumor responses. Here, we found, however, that CCL25, the only chemokine for CCR9+ cells, is not expressed in human or murine triple-negative breast cancers (TNBCs), raising a hypothesis that intratumoral delivery of CCL25 may enhance antitumor immunotherapy in TNBCs. We first determined whether this approach can enhance CD47-targeted immunotherapy using a tumor acidity-responsive nanoparticle delivery system (NP-siCD47/CCL25) to sequentially release CCL25 protein and CD47 small interfering RNA in tumor. NP-siCD47/CCL25 significantly increased infiltration of CCR9+CD8+ T cells and down-regulated CD47 expression in tumor, resulting in inhibition of tumor growth and metastasis through a T cell-dependent immunity. Furthermore, the antitumor effect of NP-siCD47/CCL25 was synergistically enhanced when used in combination with programmed cell death protein-1/programmed death ligand-1 blockades. This study offers a strategy to enhance immunotherapy by promoting CCR9+CD8+ T cell tumor infiltration.

Lentiviral gene transfer to reduce atherosclerosis progression by long-term CC-chemokine inhibition.

CC-chemokines are important mediators in the pathogenesis of atherosclerosis. Atherosclerosis progression is reduced by high-level, short-term inhibition of CC-chemokine activity, for example by adenoviral gene transfer. However, atherosclerosis is a chronic condition where short-term effects, while demonstrating proof-of-principle, are unlikely to provide maximum therapeutic benefit. Accordingly, we generated a recombinant lentivirus, lenti35K, encoding the broad-spectrum CC chemokine inhibitor, 35K, derived from the vaccinia virus. To investigate the effects of prolonged broad-spectrum chemokine inhibition on atherosclerosis, lenti35K, or lentiGFP or PBS were delivered to 6-week-old ApoE knockout (ApoE-KO) mice by hydrodynamic injection. Sustained lentiviral transduction and transgene expression were demonstrated by 35K mRNA and viral DNA in liver tissue, and recombinant 35K protein circulating in the plasma, 3 months after gene transfer. Plasma from lenti35K animals had reduced chemokine activity compared with plasma from lentiGFP or PBS-treated animals. Histologic analysis of aortic sinus sections revealed that atherosclerotic plaque area in lenti35K mice was significantly reduced compared with both lentiGFP and PBS controls. Furthermore, plaque macrophage content was substantially reduced in lenti35K mice. Lentiviral 35K gene transfer is a promising experimental strategy to reduce atherosclerosis progression, and demonstrates the potential of long-term CC-chemokine inhibition as a potential therapeutic target in atherosclerosis.

Short hairpin RNAs against eotaxin or interleukin-5 decrease airway eosinophilia and hyper-responsiveness in a murine model of asthma.

BACKGROUND: Eosinophilia plays the major role in the pathogenesis of asthma and correlates with the up-regulation of eotaxin, which, together with interleukin (IL)-5, is important for differentiation, chemo-attraction, degranulation, and survival of eosinophils in local tissue. In a previous study, we found that administration of lentivirus-delivered short hairpin RNA (shRNA) to suppress the expression of IL-5 inhibited airway inflammation. The present study aimed to investigate the role of eotaxin shRNA and the synergistic effect of eotaxin and IL-5 shRNAs on airway inflammation in an ovalbumin (OVA)-induced murine model of asthma. METHODS: Lentivirus-delivered shRNAs were used to suppress the expression of eotaxin and/or IL-5 in local tissue in an OVA-induced murine asthma model. RESULTS: Intra-tracheal administration of lentivirus containing eotaxin shRNA expressing cassette (eoSEC3.3) efficiently moderated the characteristics of asthma, including airway hyper-responsiveness, cellular infiltration of lung tissues, and eotaxin and IL-5 levels in bronchio-alveolar lavage fluid. Administration of lentiviruses expressing IL-5 or eotaxin shRNAs (IL5SEC4 + eoSEC3.3) also moderated the symptoms of asthma in a mouse model. CONCLUSIONS: Local delivery of lentiviruses expressing IL-5 and eotaxin shRNAs provides a potential tool in moderating airway inflammation and also has the potential for developing clinical therapy based on the application of shRNAs of chemokines and cytokines involved in T helper 2 cell inflammation and eosinophilia.

CCL18 enhances migration, invasion and EMT by binding CCR8 in bladder cancer cells.

Increased expression of CCL18 has been observed in various malignancies and in the urine samples of patients with bladder cancer (BC). However, the roles of CCL18 in the development, progression and metastasis of BC remain unclear. The present study demonstrated that CCL18 expression was significantly associated with advanced clinical stages of BC. Furthermore, exogenous CCL18 promoted cell invasion and migration, and induced cell epithelial­mesenchymal transition (EMT) in BC cells. Western blotting demonstrated that E­cadherin, an epithelial marker, was decreased, whereas matrix metalloproteinase (MMP)­2 and vascular endothelial growth factor (VEGF)­C were increased in CCL18­treated cells. Blocking CCR8 via a small molecule inhibitor or short hairpin (sh)RNA mitigated the decrease in E­cadherin, and increase in MMP­2 and VEGF­C, caused by human recombinant (r)CCL18. CCR8 knockdown by shRNA reversed rCCL18­induced cancer cell invasion, migration and EMT. In conclusion, these data suggested that CCL18 may promote migration, invasion and EMT by binding CCR8 in BC cells. Inhibition of CCL18 activity by blocking CCR8 could be a potential therapeutic strategy for preventing the progression of BC.

Discovery of CCL18 antagonist blocking breast cancer metastasis.

Our previous studies have proved that CCL18 is the most secreted chemokine in breast cancer microenvironment by tumor associated macrophages (TAMs). CCL18 promotes breast cancer invasiveness by binding to its cognate receptor PITPNM3 and activating the downstream signaling pathways. The high level of CCL18 in serum or tumor stroma is associated with tumor metastasis and poor patients overall survival. In this study, we identify an effective small molecular compound (SMC) to antagonize the effect of CCL18. We screen more than 1000 SMCs from Sun Yat-sen University SMC library and select 15 top scored SMCs by using computer-aided virtual screening based on the structure of CCL18. Then in vitro cell migration assay narrows down the selected 15 SMCs to the most effective SMC-21598. We find 10 µM SMC-21598 significantly inhibits CCL18-induced breast cancer cells adherence, invasiveness, and migration. Our further surface plasmon resonance (SPR), fluorescence spectroscopy and isothermal titration calorimetry (ITC) assays reveal that SMC-21598 binds tightly to CCL18, which blocks the binding of CCL18 with its receptor PITPNM3. The in vivo animal experiments show that SMC-21598 doesn't significantly affect xenografts growth, but inhibits lung metastasis. Our study provides a potential lead compound to antagonize CCL18 function. It would be of great significance to develop SMC drugs to ameliorate breast cancer metastasis and prolong patients' survival.

An intracellular allosteric site for a specific class of antagonists of the CC chemokine G protein-coupled receptors CCR4 and CCR5.

A novel mechanism for antagonism of the human chemokine receptors CCR4 and CCR5 has been discovered with a series of small-molecule compounds that seems to interact with an allosteric, intracellular site on the receptor. The existence of this site is supported by a series of observations: 1) intracellular access of these antagonists is required for their activity; 2) specific, saturable binding of a radiolabeled antagonist requires the presence of CCR4; and 3) through engineering receptor chimeras by reciprocal transfer of C-terminal domains between CCR4 and CCR5, compound binding and the selective structure-activity relationships for antagonism of these receptors seem to be associated with the integrity of that intracellular region. Published antagonists from other chemical series do not seem to bind to the novel site, and their interaction with either CCR4 or CCR5 is not affected by alteration of the C-terminal domain. The precise location of the proposed binding site remains to be determined, but the known close association of the C-terminal domain, including helix 8, as a proposed intracellular region that interacts with transduction proteins (e.g., G proteins and beta-arrestin) suggests that this could be a generic allosteric site for chemokine receptors and perhaps more broadly for class A G protein-coupled receptors. The existence of such a site that can be targeted for drug discovery has implications for screening assays for receptor antagonists, which would need, therefore, to consider compound properties for access to this intracellular site.

Specific remodeling of splenic architecture by cytomegalovirus.

Efficient immune defenses are facilitated by the organized microarchitecture of lymphoid organs, and this organization is regulated by the compartmentalized expression of lymphoid tissue chemokines. Mouse cytomegalovirus (MCMV) infection induces significant remodeling of splenic microarchitecture, including loss of marginal zone macrophage populations and dissolution of T and B cell compartmentalization. MCMV preferentially infected the splenic stroma, targeting endothelial cells (EC) as revealed using MCMV-expressing green fluorescent protein. MCMV infection caused a specific, but transient transcriptional suppression of secondary lymphoid chemokine (CCL21). The loss of CCL21 was associated with the failure of T lymphocytes to locate within the T cell zone, although trafficking to the spleen was unaltered. Expression of CCL21 in lymphotoxin (LT)-alpha-deficient mice is dramatically reduced, however MCMV infection further reduced CCL21 levels, suggesting that viral modulation of CCL21 was independent of LTalpha signaling. Activation of LTbeta-receptor signaling with an agonistic antibody partially restored CCL21 mRNA expression and redirected transferred T cells to the splenic T cell zone in MCMV-infected mice. These results indicate that virus-induced alterations in lymphoid tissues can occur through an LT-independent modulation of chemokine transcription, and targeting of the LT cytokine system can counteract lymphoid tissue remodeling by MCMV.

Modulation of eosinophil activation in vitro by a nicotinic receptor agonist.

Nicotinic receptor agonists decreased the infiltration of eosinophils into the lung and airways in a mouse model of asthma. To better understand the mechanisms implicated in this anti-inflammatory phenomenon, the expression of nicotinic acetylcholine receptors (nAChRs) and the effect of dimethylphenylpiperazinium (DMPP), a nonselective nAChR agonist, on human blood eosinophils were studied. The expression of alpha-3, -4, and -7 nAChR subunits on human blood eosinophils was measured by cell ELISA and immunocytochemistry. mRNA expression for all three subunits was evaluated by quantitative RT-PCR. The effect of DMPP on leukotriene C4 (LTC4) and matrix metalloproteinase-9 (MMP-9) production, eosinophil migration, and intracellular calcium mobilization was measured. The results show that the alpha-3, -4, and -7 nAChR subunits and mRNAs are expressed by blood eosinophils. In vitro treatment of these cells with various concentrations of DMPP reduced platelet-activating factor (PAF)-induced LTC4 production significantly. DMPP (160 microM) decreased eotaxin, and 5-oxo-6,8,11,14-eicosatetranoic acid induced eosinophil migration through Matrigel by 40.9% and 55.5%, respectively. This effect was reversed by the nAChR antagonist mecamylamine. In addition, DMPP reduced MMP-9 release and the inositol 1,4,5-triphosphate-dependent intracellular calcium increase provoked by PAF. Taken together, these results indicate that functional nAChRs are expressed on eosinophils and that nAChR agonists down-regulate eosinophil function in vitro. These anti-inflammatory effects could be of interest in the treatment of allergic asthma.