Molecular basis of ALK1-mediated signalling by BMP9/BMP10 and their prodomain-bound forms.

Activin receptor-like kinase 1 (ALK1)-mediated endothelial cell signalling in response to bone morphogenetic protein 9 (BMP9) and BMP10 is of significant importance in cardiovascular disease and cancer. However, detailed molecular mechanisms of ALK1-mediated signalling remain unclear. Here, we report crystal structures of the BMP10:ALK1 complex at 2.3 Å and the prodomain-bound BMP9:ALK1 complex at 3.3 Å. Structural analyses reveal a tripartite recognition mechanism that defines BMP9 and BMP10 specificity for ALK1, and predict that crossveinless 2 is not an inhibitor of BMP9, which is confirmed by experimental evidence. Introduction of BMP10-specific residues into BMP9 yields BMP10-like ligands with diminished signalling activity in C2C12 cells, validating the tripartite mechanism. The loss of osteogenic signalling in C2C12 does not translate into non-osteogenic activity in vivo and BMP10 also induces bone-formation. Collectively, these data provide insight into ALK1-mediated BMP9 and BMP10 signalling, facilitating therapeutic targeting of this important pathway.

Anaesthetic impairment of immune function is mediated via GABA(A) receptors.

BACKGROUND: GABA(A) receptors are members of the Cys-loop family of neurotransmitter receptors, proteins which are responsible for fast synaptic transmission, and are the site of action of wide range of drugs. Recent work has shown that Cys-loop receptors are present on immune cells, but their physiological roles and the effects of drugs that modify their function in the innate immune system are currently unclear. We are interested in how and why anaesthetics increase infections in intensive care patients; a serious problem as more than 50% of patients with severe sepsis will die. As many anaesthetics act via GABA(A) receptors, the aim of this study was to determine if these receptors are present on immune cells, and could play a role in immunocompromising patients. PRINCIPAL FINDINGS: We demonstrate, using RT-PCR, that monocytes express GABA(A) receptors constructed of α1, α4, ß2, γ1 and/or δ subunits. Whole cell patch clamp electrophysiological studies show that GABA can activate these receptors, resulting in the opening of a chloride-selective channel; activation is inhibited by the GABA(A) receptor antagonists bicuculline and picrotoxin, but not enhanced by the positive modulator diazepam. The anaesthetic drugs propofol and thiopental, which can act via GABA(A) receptors, impaired monocyte function in classic immunological chemotaxis and phagocytosis assays, an effect reversed by bicuculline and picrotoxin. SIGNIFICANCE: Our results show that functional GABA(A) receptors are present on monocytes with properties similar to CNS GABA(A) receptors. The functional data provide a possible explanation as to why chronic propofol and thiopental administration can increase the risk of infection in critically ill patients: their action on GABA(A) receptors inhibits normal monocyte behaviour. The data also suggest a potential solution: monocyte GABA(A) receptors are insensitive to diazepam, thus the use of benzodiazepines as an alternative anesthetising agent may be advantageous where infection is a life threatening problem.

Development and characterisation of an assay for furin activity.

Furin is a serine endoprotease that is responsible for the proteolytic processing of proteins within the secretory pathway, including cytokines, hormones, integrins, other proteases, and also pathogen-derived proteins. It is likely that the level of furin activity determines the extent of processing of these substrates. Furin is ubiquitously expressed across all tissues, at low levels, but can be induced in response to environmental cues such as hypoxia and cytokine stimulation. However, all studies to date that have investigated furin expression have been limited to analysis of furin mRNA; there has been no assay sensitive enough to quantify endogenous furin. Though activity-based assays have been described for furin-like enzyme activity, we demonstrate that these assays are dominated by the activity of other enzymes and cannot be used to approximate furin activity. A sensitive and specific assay for furin activity was therefore developed and characterised, using an antibody capture step to immobilise furin from whole cell lysates. Furin activity is quantified relative to that of recombinant active furin protein, to allow estimation of active furin protein concentration. The assay has a minimum detection limit of 0.006 nM; sensitive enough to determine the furin activity of many of the cell lines tested. The specificity of the assay was demonstrated by genetic modulation of furin expression. Furthermore, the assay was used to demonstrate that the cytokine transforming growth factor beta (TGF-ß) stimulates increased furin activity in HepG2 cells, confirming and extending previous reports that TGF-ß increases furin expression, and adding to the mounting body of evidence that cellular furin activity can be modulated.

Highly potent, orally available anti-inflammatory broad-spectrum chemokine inhibitors.

A series of 3-acylaminocaprolactams are inhibitors of chemokine-induced chemotaxis. Branching of the side chain alpha-carbon provides highly potent inhibitors of a range of CC and CXC chemokines. The most potent compound has an ED(50) of 40 pM. Selected compounds were tested in an in vivo inflammatory assay, and the best compound reduces TNF-alpha levels with an ED(50) of 0.1 microg/kg when administered by either subcutaneous injection or oral delivery.

Preventive usage of broad spectrum chemokine inhibitor NR58-3.14.3 reduces the severity of pulmonary and hepatic graft-versus-host disease.

Pulmonary graft-versus-host disease (pGVHD) is a major complication after allogeneic bone marrow transplantation (BMT), which involves donor leukocyte migration into the lung along chemokine gradients, leading to pulmonary dysfunction and respiratory insufficiency. As broad spectrum chemokine inhibitor (BSCI) NR58-3.14.3 suppresses leukocyte migration in response to various chemokines, including CCL2, CCL3, CCL5, we investigated the effects of NR58-3.14.3 on the evolution of pGVHD. Lethally irradiated B6D2F1 mice received BMT from syngeneic (B6D2F1) or allogeneic (C57BL/6) donors, and animals were treated with either NR58-3.14.3 or vehicle control from day -1 to day +14. At week 6, in allogeneic recipients that received BSCI, inflammatory cell infiltrates in the lung were decreased, and reduced histopathologic changes translated into improved pulmonary function when compared to allo-controls. Acute GVHD of the liver was also diminished, whereas no differences were seen in the gut. Alloantigen-dependent splenic T cell expansion and systemic TNF-alpha and IFN-gamma levels were comparable in NR58-3.14.3-treated animals and allo-controls. No suppressive effect of NR58-3.14.3 on CTL cytotoxicity was found, and diminished cellular infiltrates in lung and liver were most likely due to decreased migration of mononuclear cells. Therefore, novel approaches involving BSCIs may provide a promising tool in the management of pGVHD.

A new sensitive and specific enzyme-linked immunosorbent assay for IgD.

We have developed a new highly specific ELISA for IgD, and then used it to measure levels of circulating IgD in the serum of 480 un-selected patients from the East Anglia region of UK. The assay is both extremely sensitive and specific, with a minimum detected IgD concentration of 30 pg/ml and more than 10,000-fold specificity for IgD over all other human immunoglobulins. The assay shows linear dilution characteristics with both purified IgD and human serum, and spiking of purified IgD into either purified immunoglobulins or human serum shows c. 100% recovery. Furthermore, intra-assay and inter-assay coefficients of variation for repeated measurements of the same samples are below 10% and 15% respectively. Measurement of IgD levels on the un-selected patient population showed levels to range from <300 pg/ml to over 100 microg/ml, with a geometric mean of 8 microg/ml. The distribution is approximately normal after log transformation. Levels of circulating IgD were higher in men than in women. There was a significant negative correlation between levels of IgD and age in women, but not in men. Moreover, after adjustment for age and sex, there were statistically significantly higher levels of circulating IgD in male (but not female) smokers, compared to their non-smoking counterparts. These results highlight the care that needs to be taken to control for age, sex and cigarette smoking when examining levels of circulating IgD in future studies.

Broad spectrum chemokine inhibitors related to NR58-3.14.3.

The chemokine family consists of more than 50 structurally-related small proteins which signal through type 1 G-protein coupled receptors (GPCRs) to regulate a range of immune functions, with particular focus on regulating leukocyte trafficking. They have been implicated both in normal physiological leukocyte traffic, and in recruitment of leukocytes to sites of pathological inflammation. As a result, chemokine inhibitors may have useful anti-inflammatory therapeutic properties in vivo. Compounds with chemokine-inhibitory properties that have been described to date, fall into two broad categories: receptor-specific antagonists which block the action of one or a small number of related chemokines, and broad-spectrum chemokine inhibitors (BSCIs) which block leukocyte migration in response to many, if not all, chemokines simultaneously. Since many chemokines apparently show functional redundancy in vivo, the BSCI class are attractive candidates for development as anti-inflammatory therapies. Here, we review the development of BSCIs, with particular focus on the design and characterisation of non-peptide compounds. The key structural requirements for BSCI activity are discussed, together with their implications for the mechanism of BSCI action.

Broad-spectrum chemokine inhibition reduces vascular macrophage accumulation and collagenolysis consistent with plaque stabilization in mice.

BACKGROUND: A major determinant of the risk of myocardial infarction is the stability of the atherosclerotic plaque. Macrophage-rich plaques are more vulnerable to rupture, since macrophages excrete an excess of matrix-degrading enzymes over their inhibitors, reducing collagen content and thinning the fibrous cap. Several genetic studies have shown that disruption of signalling by the chemokine monocyte chemoattractant protein 1 reduced the lipid lesion area and macrophage accumulation in the vessel wall. METHODS: We have tested whether a similar reduction in macrophage accumulation could be achieved pharmacologically by treating apolipoprotein-E-deficient mice with the chemokine inhibitor NR58-3.14.3. RESULTS: Mice treated for various periods of time (from several days to 6 months) with NR58-3.14.3 (approximately 30 mg/kg/day) consistently had 30-40% fewer macrophages in vascular lesions, compared with mice treated with the inactive control NR58-3.14.4 or PBS vehicle. Similarly, cleaved collagen staining was lower in mice treated for up to 7 days, although this effect was not maintained when treatment time was extended to 12 weeks. The vascular lipid lesion area was unaffected by treatment, but total collagen I staining and smooth muscle cell number were both increased, suggesting that a shift to a more stable plaque phenotype had been achieved. CONCLUSIONS: Strategies, such as chemokine inhibition, to attenuate macrophage accumulation may therefore be useful to promote stabilization of atherosclerotic plaques.

Blockade of chemokine-induced signalling inhibits CCR5-dependent HIV infection in vitro without blocking gp120/CCR5 interaction.

BACKGROUND: Cellular infection with human immunodeficiency virus (HIV) both in vitro and in vivo requires a member of the chemokine receptor family to act as a co-receptor for viral entry. However, it is presently unclear to what extent the interaction of HIV proteins with chemokine receptors generates intracellular signals that are important for productive infection. RESULTS: In this study we have used a recently described family of chemokine inhibitors, termed BSCIs, which specifically block chemokine-induced chemotaxis without affecting chemokine ligands binding to their receptors. The BSCI termed Peptide 3 strongly inhibited CCR5 mediated HIV infection of THP-1 cells (83 +/- 7% inhibition assayed by immunofluoresence staining), but had no effect on gp120 binding to CCR5. Peptide 3 did not affect CXCR4-dependent infection of Jurkat T cells. CONCLUSION: These observations suggest that, in some cases, intracellular signals generated by the chemokine coreceptor may be required for a productive HIV infection.

Identification of 3-(acylamino)azepan-2-ones as stable broad-spectrum chemokine inhibitors resistant to metabolism in vivo.

3-(acylamino)glutarimides, a class of broad spectrum chemokine inhibitors, are rapidly hydrolyzed in serum, despite being stable in aqueous solution. Synthesis and high-performance liquid chromatography analysis of the proposed N-acyl-glutamate and -glutamine metabolites establish the enzyme-catalyzed breakdown pathways. In vitro assays suggest that despite their short half-life in vivo, the parent acylamino-glutarimides, not the ring-opened hydrolysis products, are the source of the antiinflammatory activity. Identification of this metabolic pathway has led to the development of 3-(acylamino)azepan-2-ones that are also broad spectrum chemokine inhibitors and act as stable, orally available powerful antiinflammatory agents in vivo with doses of 1 mg/kg.

Apolipoprotein E modulates clearance of apoptotic bodies in vitro and in vivo, resulting in a systemic proinflammatory state in apolipoprotein E-deficient mice.

Apolipoprotein E (apoE) is a 34-kDa glycoprotein involved in lipoprotein transport through interaction with the low-density lipoprotein receptor and related receptors. Recently, it has become clear that apoE binding to its receptors plays a role both in development and in control of the immune system. In this study, we show that apoE modulates the rate of uptake of apoptotic cells by macrophages. In vitro, apoE-deficient macrophages ingest less apoptotic thymocytes (but not latex beads) than wild-type macrophages, and this defect can be corrected by addition of exogenous apoE protein. In vivo, the number of dying macrophages is increased in a range of tissues, including lung and brain. Possibly in response to the larger numbers of persistent apoptotic bodies, the number of live macrophages in these tissues are also increased compared with those of wild-type control mice. In addition to the significant changes in macrophage population dynamics we observed, levels of the proinflammatory cytokine TNF-alpha and the positive acute phase reactant fibrinogen are also elevated in the livers from apoE-deficient mice. In contrast, neither deletion of the gene encoding the LDL receptor nor cholesterol feeding of wild-type mice affected either the number of apoptotic bodies or the number of live macrophages. We conclude that apoE deficiency results in impaired clearance of apoptotic cell remnants and a functionally relevant systemic proinflammatory condition in mice, independent of its role in lipoprotein metabolism. Any similar reduction of apoE activity in humans may contribute to the pathogenesis of a wide range of chronic diseases including atherosclerosis, dementia, and osteoporosis.

Broad-spectrum chemokine inhibition ameliorates experimental obliterative bronchiolitis.

BACKGROUND: Obliterative bronchiolitis (OB) affects over half of all long-term survivors after lung transplantation. Respiratory epithelial cell injury, peribronchial inflammation, and proliferation of fibrovascular connective tissue causing airway occlusion characterize this lesion. Several chemokines participate in experimental OB, and singular blockade is only partially effective. We hypothesized that a broad-spectrum chemokine inhibitor would be an effective intervention in preventing the progression of OB in an established heterotopic tracheal transplantation model. METHODS: Tracheas from Brown-Norway or Lewis rats were transplanted subcutaneously into Lewis recipients. Treated, allogeneic recipients received either a broad-spectrum chemokine inhibitor in its active (NR58.3.14.3) or inactive (NR58.3.14.4) form at a dose of 30 mg/kg daily. Luminal obstruction, epithelial loss, leukocytic infiltrates, and inflammatory cytokine mRNA levels were assessed in explanted tracheal samples 14 days after transplantation. RESULTS: After 14 days, allografts receiving the inactive chemokine inhibitor demonstrated marked peribronchial inflammation, near complete loss of respiratory epithelium, and extensive intraluminal proliferation of fibrovascular connective tissue, with a mean 84% +/- 5% reduction in airway lumen cross-sectional area. Isografts showed limited inflammation, with minimal loss of epithelium and luminal occlusion. Allogeneic recipients treated with the active chemokine inhibitor showed a significant preservation of respiratory epithelium, minimal peribronchial inflammation, and a marked decrease in the loss of airway cross-sectional area (23% +/- 1%) (p < 0.001). CONCLUSIONS: These findings further characterize the participation of chemokines in OB, and suggest that broad-spectrum chemokine inhibition may potentially be a useful therapeutic tool in slowing the progression of this disease.

Broad-spectrum chemokine inhibitors (BSCIs) and their anti-inflammatory effects in vivo.

Inappropriate inflammation is a component of a wide range of human diseases, including autoimmune disease, atherosclerosis, osteoporosis and Alzheimer's disease. Chemokines play an important role in orchestrating leukocyte recruitment during inflammation, and therefore represent an important target for anti-inflammatory therapies. Unfortunately, the chemokine system is complex, with about 50 ligands and 20 receptors, often acting with redundancy, making selection of appropriate specific antagonists difficult. One approach to overcoming this difficulty may be the development of broad-spectrum chemokine inhibitors (BSCIs). Here we review the present state of knowledge on BSCIs, including their activity in vitro and their anti-inflammatory effects in vivo, and discuss the future development of BSCIs as anti-inflammatory therapies for use in the clinic.

Design, synthesis, and preliminary pharmacological evaluation of N-acyl-3-aminoglutarimides as broad-spectrum chemokine inhibitors in vitro and anti-inflammatory agents in vivo.

A series of N-substituted 3-aminoglutarimides have been synthesized and tested for inhibitory activity against a range of chemokines in vitro and for suppression of lipopolysaccharide-induced inflammation in vivo. The results show that they represent the first class of small molecules with broad-spectrum chemokine inhibitory effects. Among the compounds studied, 10 (NR58,4) was the most potent, being active at doses between 5 and 15 nM in vitro and at 0.3 mg kg(-1) in vivo.