Henric Olsson

Nonradioactive methods for the assay of phosphoinositide 3-kinases and phosphoinositide phosphatases and selective detection of signaling lipids in cell and tissue extracts

We describe a novel approach to quantitation of phosphoinositides in cell extracts and in vitro enzyme-catalyzed reactions using suitably tagged and/or labeled pleckstrin homology (PH) domains as probes. Stable complexes were formed between the biotinylated target lipid and an appropriate PH domain, and phosphoinositides present in samples were detected by their ability to compete for binding to the PH domain. Complexes were detected using AlphaScreen technology or time-resolved FRET. The assay procedure was validated using recombinant PI 3-kinase gamma with diC8PtdIns(4,5)P(2) as substrate and general receptor for phosphoinositides-1 (GRP1) PH domain as a PtdIns(3,4,5)P(3)-specific probe. This PI 3-kinase assay was robust, was suitable for high-throughput screening platforms, and delivered expected IC(50) values for reference compounds. The approach is adaptable to a wide range of enzymes as demonstrated by assays of the tumor suppressor protein, PTEN, a phosphoinositide 3-phosphatase, which was measured using the same reagents but with diC8PtdIns(3,4,5)P(3) as substrate. PtdIns(3,4,5)P(3) present in lipid extracts of Swiss 3T3 and HL60 cells stimulated with platelet-derived growth factor and fMLP, respectively, was also detectable at picomole sensitivity. The versatility and general utility of this approach were demonstrated by exchanging the GRP1 PH domain for that of TAPP1 (which binds PtdIns(3,4)P(2) and not PtdIns(3,4,5)P(3)). This system was used to monitor the accumulation of PtdIns(3,4)P(2) in Swiss 3T3 cells exposed to an oxidative stress. It is therefore proposed that similar procedures should be capable of measuring any known phosphoinositide present in cell and tissue extracts or produced in kinase and phosphatase assays by using one of several well-characterized protein domains with appropriate phosphoinositide-binding specificity.

4-Anilino-6-phenyl-quinoline inhibitors of mitogen activated protein kinase-activated protein kinase 2 (MK2).

A class of inhibitors of mitogen activated protein kinase-activated kinase 2 (MK2) was discovered via high-throughput screening. This compound class demonstrates activity against the enzyme with sub-microM IC(50) values, and suppresses LPS-induced TNFalpha levels in THP-1 cells. MK2 inhibition kinetic measurements indicated mixed binding approaching non-ATP competitive inhibition.

Immunology, genetics and microbiota in the COPD pathophysiology: potential scope for patient stratification.

Chronic obstructive pulmonary disease (COPD) is characterized by sustained inflammation of the airways, leading to destruction of lung tissue and declining pulmonary function. Although smoking is the most obvious risk factor for COPD, only about 20% of smokers develop COPD and smoking cessation does not reverse progression of COPD, indicating that while smoking is an important cause or initiating factor, it is not the only driver of ongoing chronic inflammation and disease progression in COPD patients. We hypothesize that smoking-induced changes in lung microbiota, epithelial integrity and epigenetic control of gene expression result in autoantigen induction and perturbed immune regulation in genetically vunerable individuals. In our view, COPD patients may be stratified according to their immunological and inflammatory status related to specific changes in the lung microbiota (innate and adaptive immunity), presence of autoantigens (adaptive immunity: Th1-B-cell axis) and epigenetic modifications (inflammation and structural changes).

Targeting non-canonical nuclear factor-κB signalling attenuates neovascularization in a novel 3D model of rheumatoid arthritis synovial angiogenesis

Objective. Angiogenesis is crucial in RA disease progression. Lymphotoxin &bgr; receptor (LT&bgr;R)-induced activation of the non-canonical nuclear factor-&kgr;B (NF-&kgr;B) pathway via NF-&kgr;B-inducing kinase (NIK) has been implicated in this process. Consequently, inhibition of this pathway may hold therapeutic potential in RA. We describe a novel three-dimensional (3D) model of synovial angiogenesis incorporating endothelial cells (ECs), RA fibroblast-like synoviocytes (RAFLSs) and RA synovial fluid (RASF) to further investigate the contributions of NF-&kgr;B in this process. Methods. Spheroids consisting of RAFLSs and ECs were stimulated with RASF, the LT&bgr;R ligands LT&bgr; and LIGHT, or growth factor bFGF and VEGF, followed by quantification of EC sprouting using confocal microscopy and digital image analysis. Next, the effects of anginex, NIK-targeting siRNA (siNIK), LT&bgr;R–Ig fusion protein (baminercept) and a novel pharmacological NIK inhibitor were investigated. Results. RASF significantly promoted sprout formation, which was blocked by the established angiogenesis inhibitor anginex (P < 0.05). LT&bgr; and LIGHT induced significant sprouting (P < 0.05), as did bFGF/VEGF (P < 0.01). siNIK pre-treatment of ECs led to reductions in LT&bgr;R-induced vessel formation (P < 0.05). LT&bgr;R–Ig not only blocked LT&bgr;- or LIGHT-induced sprouting, but also RASF-induced sprouting (P < 0.05). The NIK inhibitor blocked angiogenesis induced by LT&bgr;, LIGHT, growth factors (P < 0.05) and RASF (P < 0.01). Conclusion. We present a novel 3D model of synovial angiogenesis incorporating RAFLSs, ECs and RASF that mimics the in vivo situation. Using this system, we demonstrate that non-canonical NF-&kgr;B signalling promotes neovascularization and show that this model is useful for dissecting relative contributions of signalling pathways in specific cell types to angiogenic responses and for testing pharmacological inhibitors of angiogenesis.

A Disintegrin and A Metalloproteinase-9 (ADAM9): A Novel Proteinase Culprit with Multifarious Contributions to COPD

Rationale: ADAMs (proteinases with a disintegrin and a metalloproteinase domain) have not been well studied in chronic obstructive pulmonary disease (COPD). Objectives: To investigate whether ADAM9 is linked to COPD in humans and mice. Methods: ADAM9 blood and lung levels were measured in patients with COPD versus control subjects, and in air‐ versus cigarette smoke (CS)‐exposed wild‐type mice. Wild‐type and Adam9−/− mice were exposed to air or CS for 1‐6 months, and COPD‐like lung pathologies were measured. Measurements and Main Results: ADAM9 staining was increased in lung epithelial cells and macrophages in smokers and even more so in patients with COPD and correlated directly with pack‐year smoking history and inversely with airflow obstruction and/or FEV1 percent predicted. Bronchial epithelial cell ADAM9 mRNA levels were higher in patients with COPD than control subjects and correlated directly with pack‐year smoking history. Plasma, BAL fluid, and sputum ADAM9 levels were similar in patients with COPD and control subjects. CS exposure increased Adam9 levels in wild‐type murine lungs. Adam9−/− mice were protected from emphysema development, small‐airway fibrosis, and airway mucus metaplasia. CS‐exposed Adam9−/− mice had reduced lung macrophage counts, alveolar septal cell apoptosis, lung elastin degradation, and shedding of vascular endothelial growth factor receptor‐2 and epidermal growth factor receptor in BAL fluid samples. Recombinant ADAM9 sheds epidermal growth factor and vascular endothelial growth factor receptors from epithelial cells to reduce activation of the Akt prosurvival pathway and increase cellular apoptosis. Conclusions: ADAM9 levels are increased in COPD lungs and linked to key clinical variables. Adam9 promotes emphysema development, and large‐ and small‐airway disease in mice. Inhibition of ADAM9 could be a therapeutic approach for multiple COPD phenotypes.

A Disintegrin and A Metalloproteinase Domain-8 (ADAM8): A Novel Protective Proteinase in COPD

RATIONALE ADAM8 (a disintegrin and metalloproteinase domain-8) is expressed by leukocytes and epithelial cells in health, but its contribution to the pathogenesis of chronic obstructive pulmonary disease (COPD) is unknown. OBJECTIVES To determine whether the expression of ADAM8 is increased in the lungs of patients with COPD and cigarette smoke (CS)-exposed mice, and whether ADAM8 promotes the development of COPD. METHODS ADAM8 levels were measured in lung, sputum, plasma, and/or BAL fluid samples from patients with COPD, smokers, and nonsmokers, and wild-type (WT) mice exposed to CS versus air. COPD-like lung pathologies were compared in CS-exposed WT versus Adam8-/- mice. MEASUREMENTS AND MAIN RESULTS ADAM8 immunostaining was reduced in macrophages, and alveolar and bronchial epithelial cells in the lungs of patients with COPD versus control subjects, and CS- versus air-exposed WT mice. ADAM8 levels were similar in plasma, sputum, and BAL fluid samples from patients with COPD and control subjects. CS-exposed Adam8-/- mice had greater airspace enlargement and airway mucus cell metaplasia than WT mice, but similar small airway fibrosis. CS-exposed Adam8-/- mice had higher lung macrophage counts, oxidative stress levels, and alveolar septal cell death rates, but lower alveolar septal cell proliferation rates and soluble epidermal growth factor receptor BAL fluid levels than WT mice. Adam8 deficiency increased lung inflammation by reducing CS-induced activation of the intrinsic apoptosis pathway in macrophages. Human ADAM8 proteolytically shed the epidermal growth factor receptor from bronchial epithelial cells to reduce mucin expression in vitro. Adam8 bone marrow chimera studies revealed that Adam8 deficiency in leukocytes and lung parenchymal cells contributed to the exaggerated COPD-like disease in Adam8-/- mice. CONCLUSIONS Adam8 deficiency increases CS-induced lung inflammation, emphysema, and airway mucus cell metaplasia. Strategies that increase or prolong ADAM8s expression in the lung may have therapeutic efficacy in COPD.

A7.14 Identification of new inhibitors of angiogenesis in a novel 3d model of rheumatoid arthritis synovial angiogenesis

Background/objective Angiogenesis contributes to rheumatoid arthritis (RA) pathogenesis, however, many models focus solely on endothelial cells (EC). We developed a 3D-spheroid model including both EC and RA fibroblast-like-synoviocytes (FLS) to study angiogenesis associated with RA and to test efficacy of several inhibitors targeting this process. Previous work demonstrated a role for the non-canonical NF-κB pathway and its main regulator, NF-kB inducing kinase (NIK) in pathological angiogenesis. Here we aim to use the 3D model to further characterise its contribution to neovascularization. Methods Human umbilical vein EC (HUVEC) were combined with RA FLS in spheroids and placed in a collagen gel. Spheroids were stimulated with RA synovial fluid (SF), growth factors (GF), LT or LIGHT. To establish NIK dependency in EC, EC were pre-transfected with a non-targeting or NIK-targeting siRNA before incorporation into spheroids. Pharmacological inhibitors were also added to determine their ability to abrogate EC sprouting induced by 10% RASF. Sprouting was imaged by confocal microscopy and quantified using the Leica QWin Plus software. Results Spheroids formed sprouts under all conditions with significant increases observed upon stimulation with LT, LIGHT, 10% RA SF (p < 0.05) and growth factors (p < 0.01), as compared to basal levels. LT and LIGHT induced sprout formation was NIK dependent as spheroids containing HUVEC transfected with NIK targeting siRNA had reductions in vessel formation (p < 0.05) as compared to controls. Anginex blocked sprout formation induced not only by growth factors, but also by that induced by RA SF (p < 0.05). Inhibitors of the non-canonical NF-kB pathway were able to attenuate cumulative sprout growth promoted by LT, LIGHT and RA SF (p < 0.05). Conclusion The 3D model is an effective tool for studying synovial angiogenesis in that it incorporates several elements essential to the process namely EC, RA FLS and immune cell factors found in RA SF. Using this system, we have further demonstrated a role for activation of the non-canonical NF-kB pathway, and its central regulator NIK, in neovascularization associated with RA. Moreover, we have shown this method to be useful for testing inhibitors of angiogenesis and found that targeting of non-canonical NF-kB signalling to be an effective method of blocking pathological angiogenesis.

A6.31 Contributions of canonical and non-canonical NF-κB signalling to LTR-driven inflammatory activation of endothelium and interaction with leukocytes

Background and objectives Sites of chronic inflammation, such as rheumatoid arthritis synovial tissue, are often characterised by the formation of tertiary lymphoid structures, which show characteristic features of lymphoid organs, like high endothelial venules (HEV) and sometimes even true germinal centres. Ligation of the lymphotoxin (LT)-β receptor (LTβR) results in activation of both canonical and non-canonical NF-κB signalling in endothelial cells (ECs) and plays a crucial role in lymphoid neogenesis. Non-canonical NF-κB signalling in ECs promotes inflammation-induced angiogenesis and triggers the development of the cuboidal HEV appearance. However, the relative contribution of the individual pathways to the acquisition of leukocyte traffic-regulating properties by ECs is less well understood. We therefore aimed to identify the molecular pathways by which LTβR drives activation of ECs and interactions with leukocytes. Methods Primary human ECs were stimulated with LTβ or LIGHT to activate LTβR. Activation of downstream signalling pathways was assessed by western blot analysis. Levels of cytokines secreted by LTβ-treated ECs were measured in conditioned media using antibody arrays. To block canonical NF-κB signalling pathway, a small molecule inhibitor of IKKβ was used. Inactivation of non-canonical NF-κB signalling was obtained with a small molecule inhibitor of NIK, and siRNAs targeting NIK or NFκB2. EC interactions with leukocytes were determined by an adhesion assay, whereas EC monolayer permeability was assessed by a permeability assay. Results Treatment of ECs with LTβ or LIGHT activated both canonical and non-canonical NF-κB signalling pathways. Exposure of ECs to LTβ induced the expression of several inflammatory cytokines (IL-8, IL-6, CXCL1, CXCL5, MCP-1, GM-CSF, MIP3α), and this response was mediated mainly by the canonical NF-κB pathway. Consistent with the inflammatory activation of ECs, LTβR ligation induced adhesion of T cells and monocytes to activated endothelium in a predominantly canonical NF-κB-dependent manner. Of note, LTβ was more potent at inducing adhesive properties of ECs than LIGHT. Finally, LTβR ligation by LTβ induced permeability across EC monolayers. Conclusions LTβR ligation on ECs drives inflammation-induced angiogenesis and differentiation into HEV via the non-canonical NF-κB pathway, as well as inflammatory activation of the endothelium and leukocyte adhesion through canonical NF-κB signalling, implicating ECs as immune cell mobilizers during the formation of tertiary lymphoid structures in the course of autoimmune diseases. Further molecular dissection of these pathways may help to better understand the function of tertiary lymphoid structures and potentially to develop therapeutics that modulate the formation of these structures and reduce inflammation.