Peter J. Gough

Stimulated Shedding of Vascular Cell Adhesion Molecule 1 (VCAM-1) Is Mediated by Tumor Necrosis Factor-α-converting Enzyme (ADAM 17)

A variety of cell surface adhesion molecules can exist as both transmembrane proteins and soluble circulating forms. Increases in the levels of soluble adhesion molecules have been correlated with a variety of inflammatory diseases, suggesting a pathological role. Although soluble forms are thought to result from proteolytic cleavage from the cell surface, relatively little is known about the proteases responsible for their release. In this report we demonstrate that under normal culture conditions, cells expressing vascular cell adhesion molecule 1 (VCAM-1) release a soluble form of the extracellular domain that is generated by metalloproteinase-mediated cleavage. VCAM-1 release can be rapidly simulated by phorbol 12-myristate 13-acetate (PMA), and this induced VCAM-1 shedding is mediated by metalloproteinase cleavage of VCAM-1 near the transmembrane domain. PMA-induced VCAM-1 shedding occurs as the result of activation of a specific pathway, as the generation of soluble forms of three other adhesion molecules, E-selectin, platelet-endothelial cell adhesion molecule 1, and intercellular adhesion molecule 1, are not altered by PMA stimulation. Using cells derived from genetically deficient mice, we identify tumor necrosis factor-α-converting enzyme (TACE or ADAM 17) as the protease responsible for PMA-induced VCAM-1 release, including shedding of endogenously expressed VCAM-1 by murine endothelial cells. Therefore, TACE-mediated shedding of VCAM-1 may be important for the regulation of VCAM-1 function at the cell surface.

Macrophage Class A Scavenger Receptor-Mediated Phagocytosis of Escherichia coli: Role of Cell Heterogeneity, Microbial Strain, and Culture Conditions In Vitro

ABSTRACT Macrophage class A scavenger receptors (SR-AI and SR-AII) contribute to host defense by binding polyanionic ligands such as lipopolysaccharide and lipoteichoic acid. SR-A knockout (SR-A−/−) mice are more susceptible to endotoxic shock and Listeria monocytogenes infection in vivo, possibly due to decreased clearance of lipopolysaccharide and microorganisms, respectively. We have used flow cytometry to analyze the role of SR-A and other scavenger-like receptors in phagocytosis of bacteria in vitro. Chinese hamster ovary cells stably transfected with human SR-A bound Escherichia coli and Staphylococcus aureus but ingested few organisms. Primary human monocyte-derived macrophages (Mφ) bound and ingested E. coli more efficiently, and this was partially but selectively blocked by the general SR inhibitor, poly(I). A specific and selective role for SR-A was shown, since bone marrow culture-derived Mφ from SR-A−/− mice ingested fewer E. coli organisms than did wild-type cells, while uptake of antibody-opsonized E. coli was unaffected. SR-A-dependent uptake of E. colivaried with the bacterial strain; ingestion of DH5α and K1 by SR-A−/− Mφ was reduced by 30 to 60% and 70 to 75%, respectively. Phagocytosis and endocytosis via SR-A were markedly down-modulated when Mφ were plated on serum-coated tissue culture plastic compared to bacteriologic plastic, where cell adhesion is mediated by SR-A and CR3, respectively. This paper demonstrates that SR-A can bind and ingest bacteria directly, consistent with a role in host defense in vivo, and highlights the importance of the source of the Mφ, bacterial strain, and culture conditions on receptor function in vitro.

The role of scavenger receptors in the innate immune system

Akey aspect of the innate immune system is the ability to discriminate between self and infectious nonself. This is achieved through pattern recognition receptors which directly recognise molecular epitopes expressed by microbes. Scavenger receptors (SRs) have been studied primarily due to their ability to bind and internalise modified lipoproteins, suggesting an important role in foam cell formation and the pathogenesis of atherosclerosis. However, the ability of some SRs to function as pattern recognition receptors through their binding of a wide variety of pathogens indicates a potential role in host defence. This review will detail our current understanding of the function of SRs in innate immunity, and in the initiation of aquired immune responses.

Analysis of Macrophage Scavenger Receptor (SR-A) Expression in Human Aortic Atherosclerotic Lesions

The class A scavenger receptors (SR-As) are trimeric, integral membrane glycoproteins that exhibit unusually broad ligand-binding properties. A number of studies have suggested that these receptors may play an important role in host defense and in many macrophage-associated pathological processes, including atherosclerosis and Alzheimers disease. The study of the expression and function of these receptors in human disease has been hampered by the lack of suitable antibodies recognizing human SR-A. This has generated questions regarding the nature of receptors responsible for scavenger receptor activity detected in a variety of cell types, including monocytes, macrophages, smooth muscle cells, and endothelial cells. To address these questions, we have produced high-titer antisera recognizing human SR-A by using mice deficient for SR-A (SR-A -/-). We show that SR-A -/- mice produce a significantly higher-titer immune response than do wild-type (SR-A +/+) littermates, with antisera of the former having a broad species reactivity and recognizing SR-A from humans, mice, and rabbits. The antisera recognize both type I and II SR-A in a wide range of immunological techniques. Using these antisera we show that the expression of SR-A protein is induced during monocyte to macrophage differentiation and that SR-A mediates 80% of the uptake of acetylated low density lipoprotein by human monocyte-derived macrophages. We also establish that human SR-A is expressed by tissue macrophages in liver and lung and by macrophage-derived foam cells within aortic atherosclerotic lesions, with little detectable expression by smooth muscle cells or aortic endothelium.

RECENT PROGRESS IN DEFINING THE ROLE OF SCAVENGER RECEPTORS IN LIPID TRANSPORT, ATHEROSCLEROSIS AND HOST DEFENCE

Scavenger receptors bind and internalize modified lipoproteins. There are several different classes of scavenger receptors in mammalian cells and their relative contribution to lipid transport in normal physiology and pathological conditions such as atherosclerosis has been the subject of intense investigation. Mice with a disruption in the macrophage scavenger receptor SR-A gene exhibit a reduced size of atherosclerotic lesions and also exhibit an enhanced susceptibility to pathogens and endotoxic shock. In addition to their role in lipid transport, scavenger receptors play important roles in host defence and in the regulation of acquired immunity. Recent progress in delineating the mechanisms by which oxidized LDL effects changes in gene expression will be reviewed.

The use of human CD68 transcriptional regulatory sequences to direct high-level expression of class A scavenger receptor in macrophages in vitro and in vivo

Macrophages (Mφ) play a key role in innate and acquired immunity. The study of Mφ biology has been hampered by the absence of suitable gene regulatory sequences for the overexpression of heterologous genes in Mφ. The human CD68 gene encodes a glycoprotein that is expressed in monocytes and Mφ, and therefore represents an attractive candidate gene for the generation of a Mφ‐specific gene‐targeting vector. A transgene expression cassette that combines 2·9 kb of CD68 5′ flanking sequence with the 83‐bp first intron (IVS‐1) of the CD68 gene, directed high‐level, long‐lasting expression of class A human scavenger receptor (hSR‐A) isoforms in the murine Mφ cell line, RAW‐264. By using this CD68 expression cassette to generate Mφ cell lines that overexpress a soluble secreted form of the extracellular portion of type I human SR‐A, we were able to purify significant quantities of this protein and show its ability to inhibit SR‐A‐mediated endocytosis. Analysis of two independent lines of transgenic mice that expressed type III human SR‐A under the control of the CD68 gene sequences revealed transgene mRNA expression in elicited Mφ populations and in mouse tissues in a pattern that was consistent with Mφ‐specific gene targeting. These data show that CD68 transcriptional regulatory sequences can be used to direct high‐level transgene expression in Mφin vitro and in vivo.

Autolytic Proteolysis within the Function to Find Domain (FIIND) Is Required for NLRP1 Inflammasome Activity

Background: NLRP1 mediates the release of the inflammatory cytokine IL-1β and is linked to several human inflammatory diseases. Results: Autolytic proteolysis occurs within the C terminus of NLRP1 and is modulated by polymorphisms and alternative mRNA splicing. Conclusion: Autolytic cleavage is a key regulator of the NLRP1 inflammasome and downstream IL-1β production. Significance: Understanding the mechanisms underlying NLRP1 activation is required to develop effective therapeutics. Nucleotide-binding domain leucine-rich repeat proteins (NLRs) play a key role in immunity and disease through their ability to modulate inflammation in response to pathogen-derived and endogenous danger signals. Here, we identify the requirements for activation of NLRP1, an NLR protein associated with a number of human pathologies, including vitiligo, rheumatoid arthritis, and Crohn disease. We demonstrate that NLRP1 activity is dependent upon ASC, which associates with the C-terminal CARD domain of NLRP1. In addition, we show that NLRP1 activity is dependent upon autolytic cleavage at Ser1213 within the FIIND. Importantly, this post translational event is dependent upon the highly conserved distal residue His1186. A disease-associated single nucleotide polymorphism near His1186 and a naturally occurring mRNA splice variant lacking exon 14 differentially affect this autolytic processing and subsequent NLRP1 activity. These results describe key molecular pathways that regulate NLRP1 activity and offer insight on how small sequence variations in NLR genes may influence human disease pathogenesis.

Discovery of Small Molecule RIP1 Kinase Inhibitors for the Treatment of Pathologies Associated with Necroptosis.

Potent inhibitors of RIP1 kinase from three distinct series, 1-aminoisoquinolines, pyrrolo[2,3-b]pyridines, and furo[2,3-d]pyrimidines, all of the type II class recognizing a DLG-out inactive conformation, were identified from screening of our in-house kinase focused sets. An exemplar from the furo[2,3-d]pyrimidine series showed a dose proportional response in protection from hypothermia in a mouse model of TNFα induced lethal shock.

Pharmacological Inhibition of C-C Chemokine Receptor 2 Decreases Macrophage Infiltration in the Aortic Root of the Human C-C Chemokine Receptor 2/Apolipoprotein E −/− Mouse: Magnetic Resonance Imaging Assessment

Purpose—This study assessed the pharmacological effect of a novel selective C-C chemokine receptor (CCR) 2 antagonist (GSK1344386B) on monocyte/macrophage infiltration into atherosclerotic plaque using magnetic resonance imaging (MRI) in an atherosclerotic mouse model. Methods and Results—Apolipoprotein E−/− mice expressing human CCR2 were fed a Western diet (vehicle group) or a Western diet plus10 mg/kg per day of GSK1344386B (GSK1344386B group). After the baseline MRI, mice were implanted with osmotic pumps containing angiotensin II, 1000 ng/kg per minute, to accelerate lesion formation. After five weeks of angiotensin II administration, mice received ultrasmall superparamagnetic iron oxide, an MRI contrast agent for the assessment of monocyte/macrophage infiltration to the plaque, and underwent imaging. After imaging, mice were euthanized, and the heart and aorta were harvested for ex vivo MRI and histopathological examination. After 5 weeks of dietary dosing, there were no significant differences between groups in body or liver weight or plasma cholesterol concentrations. An in vivo MRI reflected a decrease in ultrasmall superparamagnetic iron oxide contrast agent uptake in the aortic arch of the GSK1344386B group (P<0.05). An ex vivo MRI of the aortic root also reflected decreased ultrasmall superparamagnetic iron oxide uptake in the GSK1344386B group and was verified by absolute iron analysis (P<0.05). Although there was no difference in aortic root lesion area between groups, there was a 30% reduction in macrophage area observed in the GSK1344386B group (P<0.05). Conclusion—An MRI was used to noninvasively assess the decreased macrophage content in the atherosclerotic plaque after selective CCR2 inhibition.

Immunophenotyping of macrophages in human pulmonary tuberculosis and sarcoidosis

Summary.  Classic studies of tuberculosis (TB) revealed morphologic evidence of considerable heterogeneity of macrophages (MØs), but the functional significance of this heterogeneity remains unknown. We have used newly available specific antibodies for selected membrane and secretory molecules to examine the phenotype of MØs in situ in a range of South African patients with TB, compared with sarcoidosis. Patients were human immunodeficiency virus‐negative adults and children, and the examined biopsy specimens included lung and lymph nodes. Mature pulmonary MØs (alveolar, interstitial, epithelioid and multinucleated giant cells) selectively expressed scavenger receptor type A and a novel carboxypeptidase‐like antigen called carboxypeptidase‐related vitellogenin‐like MØ molecule (CPVL). CPVL did not display enhanced expression in sarcoidosis, vs. TB patients, as observed with angiotensin‐converting enzyme (ACE), a related molecule. Immunocytochemical studies with surfactant proteins (SP)‐A and ‐D showed that type II alveolar cells expressed these collectins, as did MØs, possibly after binding of secreted proteins. Studies with an antibody specific for the C‐terminus of fractalkine, a tethered CX3C chemokine, confirmed synthesis of this molecule by bronchiolar epithelial cells and occasional endothelial cells. These studies provide new marker antigens and extend previous studies on MØ differentiation, activation and local interactions in chronic human granulomatous inflammation in the lung.