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Uribe-Querol E, Rosales C. Phagocytosis. Methods Mol Biol 2024; 2813:39-64. [PMID: 38888769 DOI: 10.1007/978-1-0716-3890-3_3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/20/2024]
Abstract
One hundred years have passed since the death of Élie Metchnikoff (1845-1916). He was the first to observe the uptake of particles by cells and realized the importance of this process, named phagocytosis, for the host response to injury and infection. He also was a strong advocate of the role of phagocytosis in cellular immunity, and with this, he gave us the basis for our modern understanding of inflammation and the innate immune response. Phagocytosis is an elegant but complex process for the ingestion and elimination of pathogens, but it is also important for the elimination of apoptotic cells and hence fundamental for tissue homeostasis. Phagocytosis can be divided into four main steps: (i) recognition of the target particle, (ii) signaling to activate the internalization machinery, (iii) phagosome formation, and (iv) phagolysosome maturation. In this chapter, we present a general view of our current knowledge on phagocytosis performed mainly by professional phagocytes through antibody and complement receptors and discuss aspects that remain incompletely understood.
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Affiliation(s)
- Eileen Uribe-Querol
- Laboratorio de Biología del Desarrollo, División de Estudios de Posgrado e Investigación, Facultad de Odontología, Universidad Nacional Autónoma de México, Ciudad de México, Mexico
| | - Carlos Rosales
- Departamento de Inmunología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Ciudad de México, Mexico.
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2
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Nobiletti N, Liu J, Glading AJ. KRIT1-mediated regulation of neutrophil adhesion and motility. FEBS J 2023; 290:1078-1095. [PMID: 36107440 PMCID: PMC9957810 DOI: 10.1111/febs.16627] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/18/2022] [Revised: 08/31/2022] [Accepted: 09/12/2022] [Indexed: 12/01/2022]
Abstract
Loss of Krev interaction-trapped-1 (KRIT1) expression leads to the development of cerebral cavernous malformations (CCM), a disease in which abnormal blood vessel formation compromises the structure and function of the blood-brain barrier. The role of KRIT1 in regulating endothelial function is well-established. However, several studies have suggested that KRIT1 could also play a role in regulating nonendothelial cell types and, in particular, immune cells. In this study, we generated a mouse model with neutrophil-specific deletion of KRIT1 in order to investigate the effect of KRIT1 deficiency on neutrophil function. Neutrophils isolated from adult Ly6Gtm2621(cre)Arte Krit1flox/flox mice had a reduced ability to attach and spread on the extracellular matrix protein fibronectin and exhibited a subsequent increase in migration. However, adhesion to and migration on ICAM-1 was unchanged. In addition, we used a monomeric, fluorescently-labelled fragment of fibronectin to show that integrin activation is reduced in the absence of KRIT1 expression, though β1 integrin expression appears unchanged. Finally, neutrophil migration in response to lipopolysaccharide-induced inflammation in the lung was decreased, as shown by reduced cell number and myeloperoxidase activity in lavage samples from Krit1PMNKO mice. Altogether, we show that KRIT1 regulates neutrophil adhesion and migration, likely through regulation of integrin activation, which can lead to altered inflammatory responses in vivo.
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Affiliation(s)
- Nicholas Nobiletti
- Department of Pharmacology and Physiology, School of Medicine and Dentistry, University of Rochester, NY, USA
| | - Jing Liu
- Department of Pharmacology and Physiology, School of Medicine and Dentistry, University of Rochester, NY, USA
- Department of Pediatrics, School of Medicine and Dentistry, University of Rochester, NY, USA
| | - Angela J. Glading
- Department of Pharmacology and Physiology, School of Medicine and Dentistry, University of Rochester, NY, USA
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3
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Lalnunthangi A, Dakpa G, Tiwari S. Multifunctional role of the ubiquitin proteasome pathway in phagocytosis. PROGRESS IN MOLECULAR BIOLOGY AND TRANSLATIONAL SCIENCE 2023; 194:179-217. [PMID: 36631192 DOI: 10.1016/bs.pmbts.2022.06.023] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/14/2023]
Abstract
Phagocytosis is a specialized form of endocytosis where large cells and particles (>0.5μm) are engulfed by the phagocytic cells, and ultimately digested in the phagolysosomes. This process not only eliminates unwanted particles and pathogens from the extracellular sources, but also eliminates apoptotic cells within the body, and is critical for maintenance of tissue homeostasis. It is believed that both endocytosis and phagocytosis share common pathways after particle internalization, but specialized features and differences between these two routes of internalization are also likely. The recruitment and removal of each protein/particle during the maturation of endocytic/phagocytic vesicles has to be tightly regulated to ensure their timely action. Ubiquitin proteasome pathway (UPP), degrades unwanted proteins by post-translational modification of proteins with chains of conserved protein Ubiquitin (Ub), with subsequent recognition of Ub chains by the 26S proteasomes and substrate degradation by this protease. This pathway utilizes different Ub linkages to modify proteins to regulate protein-protein interaction, localization, and activity. Due to its vast number of targets, it is involved in many cellular pathways, including phagocytosis. This chapters describes the basic steps and signaling in phagocytosis and different roles that UPP plays at multiple steps in regulating phagocytosis directly, or through its interaction with other phagosomal proteins. How aberrations in UPP function affect phagocytosis and their association with human diseases, and how pathogens exploit this pathway for their own benefit is also discussed.
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Affiliation(s)
| | | | - Swati Tiwari
- School of Biotechnology, Jawaharlal Nehru University, New Delhi, India.
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4
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Lee M, Du H, Winer DA, Clemente-Casares X, Tsai S. Mechanosensing in macrophages and dendritic cells in steady-state and disease. Front Cell Dev Biol 2022; 10:1044729. [PMID: 36467420 PMCID: PMC9712790 DOI: 10.3389/fcell.2022.1044729] [Citation(s) in RCA: 33] [Impact Index Per Article: 11.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/16/2022] [Accepted: 11/01/2022] [Indexed: 11/18/2022] Open
Abstract
Macrophages and dendritic cells are myeloid cells that play critical roles in immune responses. Macrophages help to maintain homeostasis through tissue regeneration and the clearance of dead cells, but also mediate inflammatory processes against invading pathogens. As the most potent antigen-presenting cells, dendritic cells are important in connecting innate to adaptive immune responses via activation of T cells, and inducing tolerance under physiological conditions. While it is known that macrophages and dendritic cells respond to biochemical cues in the microenvironment, the role of extracellular mechanical stimuli is becoming increasingly apparent. Immune cell mechanotransduction is an emerging field, where accumulating evidence suggests a role for extracellular physical cues coming from tissue stiffness in promoting immune cell recruitment, activation, metabolism and inflammatory function. Additionally, many diseases such as pulmonary fibrosis, cardiovascular disease, cancer, and cirrhosis are associated with changes to the tissue biophysical environment. This review will discuss current knowledge about the effects of biophysical cues including matrix stiffness, topography, and mechanical forces on macrophage and dendritic cell behavior under steady-state and pathophysiological conditions. In addition, we will also provide insight on molecular mediators and signaling pathways important in macrophage and dendritic cell mechanotransduction.
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Affiliation(s)
- Megan Lee
- Department of Medical Microbiology and Immunology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada
| | - Huixun Du
- Buck Institute for Research on Aging, Leonard Davis School of Gerontology, University of Southern California, Los Angeles, CA, United States
| | - Daniel A. Winer
- Division of Cellular and Molecular Biology, Diabetes Research Group, Toronto General Hospital Research Institute (TGHRI), University Health Network, Toronto, ON, Canada
- Department of Immunology, University of Toronto, Toronto, ON, Canada
- Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada
- Department of Pathology, University Health Network, Toronto, ON, Canada
- Buck Institute for Research on Aging, Novato, CA, United States
| | - Xavier Clemente-Casares
- Cancer Research Institute of Northern Alberta, University of Alberta, Edmonton, AB, Canada
- Li Ka Shing Institute of Virology, University of Alberta, Edmonton, AB, Canada
| | - Sue Tsai
- Cancer Research Institute of Northern Alberta, University of Alberta, Edmonton, AB, Canada
- Li Ka Shing Institute of Virology, University of Alberta, Edmonton, AB, Canada
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5
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Cegarra C, Cameron B, Chaves C, Dabdoubi T, Do TM, Genêt B, Roudières V, Shi Y, Tchepikoff P, Lesuisse D. An innovative strategy to identify new targets for delivering antibodies to the brain has led to the exploration of the integrin family. PLoS One 2022; 17:e0274667. [PMID: 36108060 PMCID: PMC9477330 DOI: 10.1371/journal.pone.0274667] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/20/2022] [Accepted: 09/01/2022] [Indexed: 11/19/2022] Open
Abstract
Background
Increasing brain exposure of biotherapeutics is key to success in central nervous system disease drug discovery. Accessing the brain parenchyma is especially difficult for large polar molecules such as biotherapeutics and antibodies because of the blood-brain barrier. We investigated a new immunization strategy to identify novel receptors mediating transcytosis across the blood-brain barrier.
Method
We immunized mice with primary non-human primate brain microvascular endothelial cells to obtain antibodies. These antibodies were screened for their capacity to bind and to be internalized by primary non-human primate brain microvascular endothelial cells and Human Cerebral Microvascular Endothelial Cell clone D3. They were further evaluated for their transcytosis capabilities in three in vitro blood-brain barrier models. In parallel, their targets were identified by two different methods and their pattern of binding to human tissue was investigated using immunohistochemistry.
Results
12 antibodies with unique sequence and internalization capacities were selected amongst more than six hundred. Aside from one antibody targeting Activated Leukocyte Cell Adhesion Molecule and one targeting Striatin3, most of the other antibodies recognized β1 integrin and its heterodimers. The antibody with the best transcytosis capabilities in all blood-brain barrier in vitro models and with the best binding capacity was an anti-αnβ1 integrin. In comparison, commercial anti-integrin antibodies performed poorly in transcytosis assays, emphasizing the originality of the antibodies derived here. Immunohistochemistry studies showed specific vascular staining on human and non-human primate tissues.
Conclusions
This transcytotic behavior has not previously been reported for anti-integrin antibodies. Further studies should be undertaken to validate this new mechanism in vivo and to evaluate its potential in brain delivery.
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Affiliation(s)
- Céline Cegarra
- Rare and Neurologic Diseases Research Therapeutic Area, Sanofi, Chilly Mazarin, France
- * E-mail:
| | | | - Catarina Chaves
- Rare and Neurologic Diseases Research Therapeutic Area, Sanofi, Chilly Mazarin, France
| | | | - Tuan-Minh Do
- Rare and Neurologic Diseases Research Therapeutic Area, Sanofi, Chilly Mazarin, France
| | - Bruno Genêt
- Integrated Drug Discovery, Sanofi, Vitry-Sur-Seine, France
| | - Valérie Roudières
- Rare and Neurologic Diseases Research Therapeutic Area, Sanofi, Chilly Mazarin, France
| | - Yi Shi
- Histology, Translational Sciences, Sanofi, Vitry-Sur-Seine, France
| | | | - Dominique Lesuisse
- Rare and Neurologic Diseases Research Therapeutic Area, Sanofi, Chilly Mazarin, France
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6
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Gahmberg CG, Grönholm M, Madhavan S. Regulation of Dynamic Cell Adhesion by Integrin-Integrin Crosstalk. Cells 2022; 11:cells11101685. [PMID: 35626722 PMCID: PMC9140058 DOI: 10.3390/cells11101685] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/21/2022] [Revised: 05/11/2022] [Accepted: 05/17/2022] [Indexed: 02/07/2023] Open
Abstract
Most cells express several integrins. The integrins are able to respond to various cellular functions and needs by modifying their own activation state, but in addition by their ability to regulate each other by activation or inhibition. This crosstalk or transdominant regulation is strictly controlled. The mechanisms resulting in integrin crosstalk are incompletely understood, but they often involve intracellular signalling routes also used by other cell surface receptors. Several studies show that the integrin cytoplasmic tails bind to a number of cytoskeletal and adaptor molecules in a regulated manner. Recent work has shown that phosphorylations of integrins and key intracellular molecules are of pivotal importance in integrin-cytoplasmic interactions, and these in turn affect integrin activity and crosstalk. The integrin β-chains play a central role in regulating crosstalk. In addition to Integrin-integrin crosstalk, crosstalk may also occur between integrins and related receptors, including other adhesion receptors, growth factor and SARS-CoV-2 receptors.
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Affiliation(s)
- Carl G. Gahmberg
- Molecular and Integrative Biosciences Research Program, Faculty of Biological and Environmental Sciences, University of Helsinki, Viikinkaari 9 C, 00014 Helsinki, Finland; (M.G.); (S.M.)
- Correspondence: ; Tel.: +358-50-539-9439
| | - Mikaela Grönholm
- Molecular and Integrative Biosciences Research Program, Faculty of Biological and Environmental Sciences, University of Helsinki, Viikinkaari 9 C, 00014 Helsinki, Finland; (M.G.); (S.M.)
- Drug Research Program, Faculty of Pharmacy, University of Helsinki, Viikinkaari 9 C, 00014 Helsinki, Finland
| | - Sudarrshan Madhavan
- Molecular and Integrative Biosciences Research Program, Faculty of Biological and Environmental Sciences, University of Helsinki, Viikinkaari 9 C, 00014 Helsinki, Finland; (M.G.); (S.M.)
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Kearney KJ, Ariëns RAS, Macrae FL. The Role of Fibrin(ogen) in Wound Healing and Infection Control. Semin Thromb Hemost 2021; 48:174-187. [PMID: 34428799 DOI: 10.1055/s-0041-1732467] [Citation(s) in RCA: 27] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
Abstract
Fibrinogen, one of the most abundant plasma proteins playing a key role in hemostasis, is an important modulator of wound healing and host defense against microbes. In the current review, we address the role of fibrin(ogen) throughout the process of wound healing and subsequent tissue repair. Initially fibrin(ogen) acts as a provisional matrix supporting incoming leukocytes and acting as reservoir for growth factors. It later goes on to support re-epithelialization, angiogenesis, and fibroplasia. Importantly, removal of fibrin(ogen) from the wound is essential for wound healing to progress. We also discuss how fibrin(ogen) functions through several mechanisms to protect the host against bacterial infection by providing a physical barrier, entrapment of bacteria in fibrin(ogen) networks, and by directing immune cell function. The central role of fibrin(ogen) in defense against bacterial infection has made it a target of bacterial proteins, evolved to interact with fibrin(ogen) to manipulate clot formation and degradation for the purpose of promoting microbial virulence and survival. Further understanding of the dual roles of fibrin(ogen) in wound healing and infection could provide novel means of therapy to improve recovery from surgical or chronic wounds and help to prevent infection from highly virulent bacterial strains, including those resistant to antibiotics.
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Affiliation(s)
- Katherine J Kearney
- Discovery and Translational Science Department, Leeds Institute of Cardiovascular and Metabolic Medicine, University of Leeds, Leeds, United Kingdom
| | - Robert A S Ariëns
- Discovery and Translational Science Department, Leeds Institute of Cardiovascular and Metabolic Medicine, University of Leeds, Leeds, United Kingdom.,Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, The Netherlands
| | - Fraser L Macrae
- Discovery and Translational Science Department, Leeds Institute of Cardiovascular and Metabolic Medicine, University of Leeds, Leeds, United Kingdom
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Yoshizaki S, Tamaru T, Hara M, Kijima K, Tanaka M, Konno DJ, Matsumoto Y, Nakashima Y, Okada S. Microglial inflammation after chronic spinal cord injury is enhanced by reactive astrocytes via the fibronectin/β1 integrin pathway. J Neuroinflammation 2021; 18:12. [PMID: 33407620 PMCID: PMC7789752 DOI: 10.1186/s12974-020-02059-x] [Citation(s) in RCA: 42] [Impact Index Per Article: 10.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/16/2020] [Accepted: 12/11/2020] [Indexed: 12/23/2022] Open
Abstract
BACKGROUND After spinal cord injury (SCI), glial scarring is mainly formed around the lesion and inhibits axon regeneration. Recently, we reported that anti-β1 integrin antibody (β1Ab) had a therapeutic effect on astrocytes by preventing the induction of glial scar formation. However, the cellular components within the glial scar are not only astrocytes but also microglia, and whether or not β1Ab treatment has any influence on microglia within the glial scar remains unclear. METHODS To evaluate the effects of β1Ab treatment on microglia within the glial scar after SCI, we applied thoracic contusion SCI to C57BL/6N mice, administered β1Ab in the sub-acute phase, and analyzed the injured spinal cords with immunohistochemistry in the chronic phase. To examine the gene expression in microglia and glial scars, we selectively collected microglia with fluorescence-activated cell sorting and isolated the glial scars using laser-captured microdissection (LMD). To examine the interaction between microglia and astrocytes within the glial scar, we stimulated BV-2 microglia with conditioned medium of reactive astrocytes (RACM) in vitro, and the gene expression of TNFα (pro-inflammatory M1 marker) was analyzed via quantitative polymerase chain reaction. We also isolated both naïve astrocytes (NAs) and reactive astrocytes (RAs) with LMD and examined their expression of the ligands for β1 integrin receptors. Statistical analyses were performed using Wilcoxon's rank-sum test. RESULTS After performing β1Ab treatment, the microglia were scattered within the glial scar and the expression of TNFα in both the microglia and the glial scar were significantly suppressed after SCI. This in vivo alteration was attributed to fibronectin, a ligand of β1 integrin receptors. Furthermore, the microglial expression of TNFα was shown to be regulated by RACM as well as fibronectin in vitro. We also confirmed that fibronectin was secreted by RAs both in vitro and in vivo. These results highlighted the interaction mediated by fibronectin between RAs and microglia within the glial scar. CONCLUSION Microglial inflammation was enhanced by RAs via the fibronectin/β1 integrin pathway within the glial scar after SCI. Our results suggested that β1Ab administration had therapeutic potential for ameliorating both glial scar formation and persistent neuroinflammation in the chronic phase after SCI.
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Affiliation(s)
- Shingo Yoshizaki
- Department of Orthopedic Surgery, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582 Japan
- Department of Neuroscience & Immunology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582 Japan
| | - Tetsuya Tamaru
- Department of Orthopedic Surgery, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582 Japan
- Department of Neuroscience & Immunology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582 Japan
| | - Masamitsu Hara
- Department of Orthopedic Surgery, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582 Japan
| | - Ken Kijima
- Department of Orthopedic Surgery, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582 Japan
| | - Masatake Tanaka
- Department of Neuroscience & Immunology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582 Japan
| | - Dai-jiro Konno
- Department of Neuroscience & Immunology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582 Japan
| | - Yoshihiro Matsumoto
- Department of Orthopedic Surgery, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582 Japan
| | - Yasuharu Nakashima
- Department of Orthopedic Surgery, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582 Japan
| | - Seiji Okada
- Department of Orthopedic Surgery, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582 Japan
- Department of Neuroscience & Immunology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582 Japan
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Willmann EA, Pandurovic V, Jokinen A, Beckley D, Bohlson SS. Extracellular signal-regulated kinase 1/2 is required for complement component C1q and fibronectin dependent enhancement of Fcγ- receptor mediated phagocytosis in mouse and human cells. BMC Immunol 2020; 21:61. [PMID: 33317446 PMCID: PMC7734837 DOI: 10.1186/s12865-020-00393-6] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/11/2020] [Accepted: 12/02/2020] [Indexed: 11/25/2022] Open
Abstract
Background C1q is a soluble pattern recognition protein that regulates multiple leukocyte functions, and deficiency in C1q results in autoimmunity. C1q stimulates enhanced phagocytic function through multiple mechanisms including the rapid enhancement of Fcγ receptor (FcγR) -mediated phagocytosis. The molecular mechanism responsible for this rapid enhancement of phagocytic function is unknown. The purpose of this study was to investigate the molecular pathway required for C1q-dependent enhanced phagocytosis. Results Leukocyte associated immunoglobulin like receptor-1 (LAIR-1) is a receptor that mediates C1q-dependent activation of leukocytes; however, using LAIR-1 deficient mouse bone marrow derived macrophages (BMDM), we demonstrated that LAIR-1 was not required for C1q-dependent enhanced FcγR-mediated phagocytosis. A phospho-kinase array identified extracellular signal-regulated kinase (ERK) 1/2 as dysregulated following activation with C1q. Validation of the array in BMDM and the human monocyte cell line THP-1 demonstrated a decrease in basal ERK1/2 phosphorylation in C1q-stimulated cells compared to control cells. However, subsequent stimulation with immune complexes stimulated rapid upregulation of phosphorylation. The extracellular matrix protein fibronectin regulates enhanced phagocytic activity in macrophages similar to C1q, and both C1q and fibronectin-dependent enhanced phagocytosis required ERK1/2 since both were blocked by pharmacologic inhibition of ERK1/2. Furthermore, diminished C1q-dependent ERK1/2 phosphorylation was sustained after four-hour treatment with lipopolysaccharide and correlated with a significant reduction in TNFα production. Conclusions These data demonstrate that C1q and fibronectin utilize a similar ERK1/2-dependent mechanism for enhanced phagocytosis, which should lead to development of novel approaches to modulate C1q-dependent regulation of macrophage activation, inflammation and autoimmunity. Supplementary Information The online version contains supplementary material available at 10.1186/s12865-020-00393-6.
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Affiliation(s)
- Emily A Willmann
- Department of Microbiology and Immunology, Des Moines University, Des Moines, IA, USA
| | - Vesna Pandurovic
- Department of Microbiology and Immunology, Des Moines University, Des Moines, IA, USA
| | - Anna Jokinen
- Department of Microbiology and Immunology, Des Moines University, Des Moines, IA, USA
| | - Danielle Beckley
- Department of Microbiology and Immunology, Des Moines University, Des Moines, IA, USA
| | - Suzanne S Bohlson
- Department of Molecular Biology and Biochemistry, University of California, Irvine, CA, USA.
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10
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Lateral diffusion of CD14 and TLR2 in macrophage plasma membrane assessed by raster image correlation spectroscopy and single particle tracking. Sci Rep 2020; 10:19375. [PMID: 33168941 PMCID: PMC7652837 DOI: 10.1038/s41598-020-76272-2] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/28/2020] [Accepted: 10/23/2020] [Indexed: 01/02/2023] Open
Abstract
The diffusion of membrane receptors is central to many biological processes, such as signal transduction, molecule translocation, and ion transport, among others; consequently, several advanced fluorescence microscopy techniques have been developed to measure membrane receptor mobility within live cells. The membrane-anchored receptor cluster of differentiation 14 (CD14) and the transmembrane toll-like receptor 2 (TLR2) are important receptors in the plasma membrane of macrophages that activate the intracellular signaling cascade in response to pathogenic stimuli. The aim of the present work was to compare the diffusion coefficients of CD14 and TLR2 on the apical and basal membranes of macrophages using two fluorescence-based methods: raster image correlation spectroscopy (RICS) and single particle tracking (SPT). In the basal membrane, the diffusion coefficients obtained from SPT and RICS were found to be comparable and revealed significantly faster diffusion of CD14 compared with TLR2. In addition, RICS showed that the diffusion of both receptors was significantly faster in the apical membrane than in the basal membrane, suggesting diffusion hindrance by the adhesion of the cells to the substrate. This finding highlights the importance of selecting the appropriate membrane (i.e., basal or apical) and corresponding method when measuring receptor diffusion in live cells. Accurately knowing the diffusion coefficient of two macrophage receptors involved in the response to pathogen insults will facilitate the study of changes that occur in signaling in these cells as a result of aging and disease.
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11
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Samaržija I, Dekanić A, Humphries JD, Paradžik M, Stojanović N, Humphries MJ, Ambriović-Ristov A. Integrin Crosstalk Contributes to the Complexity of Signalling and Unpredictable Cancer Cell Fates. Cancers (Basel) 2020; 12:E1910. [PMID: 32679769 PMCID: PMC7409212 DOI: 10.3390/cancers12071910] [Citation(s) in RCA: 30] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/22/2020] [Revised: 07/10/2020] [Accepted: 07/12/2020] [Indexed: 12/12/2022] Open
Abstract
Integrins are heterodimeric cell surface receptors composed of α and β subunits that control adhesion, proliferation and gene expression. The integrin heterodimer binding to ligand reorganises the cytoskeletal networks and triggers multiple signalling pathways that can cause changes in cell cycle, proliferation, differentiation, survival and motility. In addition, integrins have been identified as targets for many different diseases, including cancer. Integrin crosstalk is a mechanism by which a change in the expression of a certain integrin subunit or the activation of an integrin heterodimer may interfere with the expression and/or activation of other integrin subunit(s) in the very same cell. Here, we review the evidence for integrin crosstalk in a range of cellular systems, with a particular emphasis on cancer. We describe the molecular mechanisms of integrin crosstalk, the effects of cell fate determination, and the contribution of crosstalk to therapeutic outcomes. Our intention is to raise awareness of integrin crosstalk events such that the contribution of the phenomenon can be taken into account when researching the biological or pathophysiological roles of integrins.
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Affiliation(s)
- Ivana Samaržija
- Laboratory for Cell Biology and Signalling, Division of Molecular Biology, Ruđer Bošković Institute, 10000 Zagreb, Croatia; (I.S.); (M.P.); (N.S.)
| | - Ana Dekanić
- Laboratory for Protein Dynamics, Division of Molecular Medicine, Ruđer Bošković Institute, 10000 Zagreb, Croatia;
| | - Jonathan D. Humphries
- Wellcome Centre for Cell-Matrix Research, Faculty of Biology, Medicine & Health, University of Manchester, Manchester M13 9PT, UK; (J.D.H.); (M.J.H.)
| | - Mladen Paradžik
- Laboratory for Cell Biology and Signalling, Division of Molecular Biology, Ruđer Bošković Institute, 10000 Zagreb, Croatia; (I.S.); (M.P.); (N.S.)
| | - Nikolina Stojanović
- Laboratory for Cell Biology and Signalling, Division of Molecular Biology, Ruđer Bošković Institute, 10000 Zagreb, Croatia; (I.S.); (M.P.); (N.S.)
| | - Martin J. Humphries
- Wellcome Centre for Cell-Matrix Research, Faculty of Biology, Medicine & Health, University of Manchester, Manchester M13 9PT, UK; (J.D.H.); (M.J.H.)
| | - Andreja Ambriović-Ristov
- Laboratory for Cell Biology and Signalling, Division of Molecular Biology, Ruđer Bošković Institute, 10000 Zagreb, Croatia; (I.S.); (M.P.); (N.S.)
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12
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Uribe-Querol E, Rosales C. Phagocytosis: Our Current Understanding of a Universal Biological Process. Front Immunol 2020; 11:1066. [PMID: 32582172 PMCID: PMC7280488 DOI: 10.3389/fimmu.2020.01066] [Citation(s) in RCA: 335] [Impact Index Per Article: 67.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/05/2020] [Accepted: 05/04/2020] [Indexed: 12/22/2022] Open
Abstract
Phagocytosis is a cellular process for ingesting and eliminating particles larger than 0.5 μm in diameter, including microorganisms, foreign substances, and apoptotic cells. Phagocytosis is found in many types of cells and it is, in consequence an essential process for tissue homeostasis. However, only specialized cells termed professional phagocytes accomplish phagocytosis with high efficiency. Macrophages, neutrophils, monocytes, dendritic cells, and osteoclasts are among these dedicated cells. These professional phagocytes express several phagocytic receptors that activate signaling pathways resulting in phagocytosis. The process of phagocytosis involves several phases: i) detection of the particle to be ingested, ii) activation of the internalization process, iii) formation of a specialized vacuole called phagosome, and iv) maturation of the phagosome to transform it into a phagolysosome. In this review, we present a general view of our current understanding on cells, phagocytic receptors and phases involved in phagocytosis.
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Affiliation(s)
- Eileen Uribe-Querol
- División de Estudios de Posgrado e Investigación, Facultad de Odontología, Universidad Nacional Autónoma de México, Mexico City, Mexico
| | - Carlos Rosales
- Departamento de Inmunología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Mexico City, Mexico
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13
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Torres-Gomez A, Cabañas C, Lafuente EM. Phagocytic Integrins: Activation and Signaling. Front Immunol 2020; 11:738. [PMID: 32425937 PMCID: PMC7203660 DOI: 10.3389/fimmu.2020.00738] [Citation(s) in RCA: 36] [Impact Index Per Article: 7.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/30/2020] [Accepted: 03/31/2020] [Indexed: 01/06/2023] Open
Abstract
Phagocytic integrins are endowed with the ability to engulf and dispose of particles of different natures. Evolutionarily conserved from worms to humans, they are involved in pathogen elimination and apoptotic and tumoral cell clearance. Research in the field of integrin-mediated phagocytosis has shed light on the molecular events controlling integrin activation and their effector functions. However, there are still some aspects of the regulation of the phagocytic process that need to be clarified. Here, we have revised the molecular events controlling phagocytic integrin activation and the downstream signaling driving particle engulfment, and we have focused particularly on αMβ2/CR3, αXβ2/CR4, and a brief mention of αVβ5/αVβ3integrins.
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Affiliation(s)
- Alvaro Torres-Gomez
- Department of Immunology, Ophthalmology and Otorhinolaryngology, School of Medicine, Universidad Complutense de Madrid, Madrid, Spain.,Instituto de Investigación Sanitaria Hospital 12 de Octubre (i+12), Madrid, Spain
| | - Carlos Cabañas
- Department of Immunology, Ophthalmology and Otorhinolaryngology, School of Medicine, Universidad Complutense de Madrid, Madrid, Spain.,Instituto de Investigación Sanitaria Hospital 12 de Octubre (i+12), Madrid, Spain.,Severo Ochoa Center for Molecular Biology (CSIC-UAM), Madrid, Spain
| | - Esther M Lafuente
- Department of Immunology, Ophthalmology and Otorhinolaryngology, School of Medicine, Universidad Complutense de Madrid, Madrid, Spain.,Instituto de Investigación Sanitaria Hospital 12 de Octubre (i+12), Madrid, Spain
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14
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Gay D, Ghinatti G, Guerrero-Juarez CF, Ferrer RA, Ferri F, Lim CH, Murakami S, Gault N, Barroca V, Rombeau I, Mauffrey P, Irbah L, Treffeisen E, Franz S, Boissonnas A, Combadière C, Ito M, Plikus MV, Romeo PH. Phagocytosis of Wnt inhibitor SFRP4 by late wound macrophages drives chronic Wnt activity for fibrotic skin healing. SCIENCE ADVANCES 2020; 6:eaay3704. [PMID: 32219160 PMCID: PMC7083618 DOI: 10.1126/sciadv.aay3704] [Citation(s) in RCA: 70] [Impact Index Per Article: 14.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 06/12/2019] [Accepted: 12/20/2019] [Indexed: 05/20/2023]
Abstract
Human and murine skin wounding commonly results in fibrotic scarring, but the murine wounding model wound-induced hair neogenesis (WIHN) can frequently result in a regenerative repair response. Here, we show in single-cell RNA sequencing comparisons of semi-regenerative and fibrotic WIHN wounds, increased expression of phagocytic/lysosomal genes in macrophages associated with predominance of fibrotic myofibroblasts in fibrotic wounds. Investigation revealed that macrophages in the late wound drive fibrosis by phagocytizing dermal Wnt inhibitor SFRP4 to establish persistent Wnt activity. In accordance, phagocytosis abrogation resulted in transient Wnt activity and a more regenerative healing. Phagocytosis of SFRP4 was integrin-mediated and dependent on the interaction of SFRP4 with the EDA splice variant of fibronectin. In the human skin condition hidradenitis suppurativa, phagocytosis of SFRP4 by macrophages correlated with fibrotic wound repair. These results reveal that macrophages can modulate a key signaling pathway via phagocytosis to alter the skin wound healing fate.
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Affiliation(s)
- Denise Gay
- CEA/DRF/IBFJ/iRCM/LRTS, 92265 Fontenay-aux-Roses cedex, France
- Inserm U1074, 92265 Fontenay-aux-Roses cedex, France
| | - Giulia Ghinatti
- CEA/DRF/IBFJ/iRCM/LRTS, 92265 Fontenay-aux-Roses cedex, France
- Inserm U1074, 92265 Fontenay-aux-Roses cedex, France
- Université Paris-Diderot, Paris 7, France
- Université Paris-Sud, Paris 11, France
| | - Christian F. Guerrero-Juarez
- Department of Developmental and Cell Biology, Sue and Bill Gross Stem Cell Research Center, NSF-Simons Center for Multiscale Cell Fate Research, Center for Complex Biological Systems, University of California, Irvine, Irvine, CA 92697, USA
| | - Rubén A. Ferrer
- Department of Dermatology, University Leipzig Medical Center, Leipzig, Germany
| | - Federica Ferri
- CEA/DRF/IBFJ/iRCM/LRTS, 92265 Fontenay-aux-Roses cedex, France
- Inserm U1074, 92265 Fontenay-aux-Roses cedex, France
- Université Paris-Diderot, Paris 7, France
- Université Paris-Sud, Paris 11, France
| | - Chae Ho Lim
- Ronald O. Perelman Department of Dermatology and Cell Biology, School of Medicine, New York University, New York, NY 10016, USA
| | - Shohei Murakami
- CEA/DRF/IBFJ/iRCM/LRTS, 92265 Fontenay-aux-Roses cedex, France
- Inserm U1074, 92265 Fontenay-aux-Roses cedex, France
- Université Paris-Diderot, Paris 7, France
- Université Paris-Sud, Paris 11, France
| | - Nathalie Gault
- CEA/DRF/IBFJ/iRCM/LRTS, 92265 Fontenay-aux-Roses cedex, France
- Inserm U1074, 92265 Fontenay-aux-Roses cedex, France
- Université Paris-Diderot, Paris 7, France
- Université Paris-Sud, Paris 11, France
| | - Vilma Barroca
- CEA/DRF/IBFJ/iRCM/LRTS, 92265 Fontenay-aux-Roses cedex, France
- Inserm U1074, 92265 Fontenay-aux-Roses cedex, France
- Université Paris-Diderot, Paris 7, France
- Université Paris-Sud, Paris 11, France
| | - Isabelle Rombeau
- Charles River Laboratories, 169 Bois des Oncins, 69210 Saint-Germain-Nuelles, France
| | - Philippe Mauffrey
- CEA/DRF/IBFJ/iRCM/LRTS, 92265 Fontenay-aux-Roses cedex, France
- Inserm U1074, 92265 Fontenay-aux-Roses cedex, France
- Université Paris-Diderot, Paris 7, France
| | - Lamya Irbah
- CEA/DRF/IBFJ/iRCM/LRTS, 92265 Fontenay-aux-Roses cedex, France
- Inserm U1074, 92265 Fontenay-aux-Roses cedex, France
- Université Paris-Diderot, Paris 7, France
| | - Elsa Treffeisen
- Department of Pediatrics, Cohen Children's Medical Center Northwell Health, New Hyde Park, NY 11040, USA
| | - Sandra Franz
- Department of Dermatology, University Leipzig Medical Center, Leipzig, Germany
- DFG-German Research Council Transregio 67, Leipzig-Dresden, Germany
| | - Alexandre Boissonnas
- Sorbonne Université, Inserm, CNRS, Centre d’Immunologie et des Maladies Infectieuses, Cimi-Paris, F-75013, Paris, France
| | - Christophe Combadière
- Sorbonne Université, Inserm, CNRS, Centre d’Immunologie et des Maladies Infectieuses, Cimi-Paris, F-75013, Paris, France
| | - Mayumi Ito
- Ronald O. Perelman Department of Dermatology and Cell Biology, School of Medicine, New York University, New York, NY 10016, USA
| | - Maksim V. Plikus
- Department of Developmental and Cell Biology, Sue and Bill Gross Stem Cell Research Center, NSF-Simons Center for Multiscale Cell Fate Research, Center for Complex Biological Systems, University of California, Irvine, Irvine, CA 92697, USA
| | - Paul-Henri Romeo
- CEA/DRF/IBFJ/iRCM/LRTS, 92265 Fontenay-aux-Roses cedex, France
- Inserm U1074, 92265 Fontenay-aux-Roses cedex, France
- Université Paris-Diderot, Paris 7, France
- Université Paris-Sud, Paris 11, France
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15
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Meli VS, Veerasubramanian PK, Atcha H, Reitz Z, Downing TL, Liu WF. Biophysical regulation of macrophages in health and disease. J Leukoc Biol 2019; 106:283-299. [PMID: 30861205 PMCID: PMC7001617 DOI: 10.1002/jlb.mr0318-126r] [Citation(s) in RCA: 91] [Impact Index Per Article: 15.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
Macrophages perform critical functions for homeostasis and immune defense in tissues throughout the body. These innate immune cells are capable of recognizing and clearing dead cells and pathogens, and orchestrating inflammatory and healing processes that occur in response to injury. In addition, macrophages are involved in the progression of many inflammatory diseases including cardiovascular disease, fibrosis, and cancer. Although it has long been known that macrophages respond dynamically to biochemical signals in their microenvironment, the role of biophysical cues has only recently emerged. Furthermore, many diseases that involve macrophages are also characterized by changes to the tissue biophysical environment. This review will discuss current knowledge about the effects of biophysical cues including matrix stiffness, material topography, and applied mechanical forces, on macrophage behavior. We will also describe the role of molecules that are known to be important for mechanotransduction, including adhesion molecules, ion channels, as well as nuclear mediators such as transcription factors, scaffolding proteins, and epigenetic regulators. Together, this review will illustrate a developing role of biophysical cues in macrophage biology, and also speculate upon molecular targets that may potentially be exploited therapeutically to treat disease.
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Affiliation(s)
- Vijaykumar S. Meli
- Department of Biomedical Engineering, University of California Irvine, CA 92697
- The Edwards Lifesciences Center for Advanced Cardiovascular Technology, University of California Irvine, CA 92697
| | - Praveen K. Veerasubramanian
- Department of Biomedical Engineering, University of California Irvine, CA 92697
- The Edwards Lifesciences Center for Advanced Cardiovascular Technology, University of California Irvine, CA 92697
| | - Hamza Atcha
- Department of Biomedical Engineering, University of California Irvine, CA 92697
- The Edwards Lifesciences Center for Advanced Cardiovascular Technology, University of California Irvine, CA 92697
| | - Zachary Reitz
- Department of Biomedical Engineering, University of California Irvine, CA 92697
- The Edwards Lifesciences Center for Advanced Cardiovascular Technology, University of California Irvine, CA 92697
| | - Timothy L. Downing
- Department of Biomedical Engineering, University of California Irvine, CA 92697
- The Edwards Lifesciences Center for Advanced Cardiovascular Technology, University of California Irvine, CA 92697
- Department of Microbiology and Molecular Genetics, University of California Irvine, CA 92697
| | - Wendy F. Liu
- Department of Biomedical Engineering, University of California Irvine, CA 92697
- The Edwards Lifesciences Center for Advanced Cardiovascular Technology, University of California Irvine, CA 92697
- Department of Chemical and Biomolecular Engineering, University of California Irvine, CA 92697
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16
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Van Agthoven JF, Shams H, Cochran FV, Alonso JL, Kintzing JR, Garakani K, Adair BD, Xiong JP, Mofrad MRK, Cochran JR, Arnaout MA. Structural Basis of the Differential Binding of Engineered Knottins to Integrins αVβ3 and α5β1. Structure 2019; 27:1443-1451.e6. [PMID: 31353240 DOI: 10.1016/j.str.2019.06.011] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/11/2019] [Revised: 05/29/2019] [Accepted: 06/28/2019] [Indexed: 01/06/2023]
Abstract
Targeting both integrins αVβ3 and α5β1 simultaneously appears to be more effective in cancer therapy than targeting each one alone. The structural requirements for bispecific binding of ligand to integrins have not been fully elucidated. RGD-containing knottin 2.5F binds selectively to αVβ3 and α5β1, whereas knottin 2.5D is αVβ3 specific. To elucidate the structural basis of this selectivity, we determined the structures of 2.5F and 2.5D as apo proteins and in complex with αVβ3, and compared their interactions with integrins using molecular dynamics simulations. These studies show that 2.5D engages αVβ3 by an induced fit, but conformational selection of a flexible RGD loop accounts for high-affinity selective binding of 2.5F to both integrins. The contrasting binding of the highly flexible low-affinity linear RGD peptides to multiple integrins suggests that a "Goldilocks zone" of conformational flexibility of the RGD loop in 2.5F underlies its selective binding promiscuity to integrins.
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Affiliation(s)
- Johannes F Van Agthoven
- Leukocyte Biology and Inflammation Program, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA 02129, USA; Structural Biology Program, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA 02129, USA; Division of Nephrology/Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA 02129, USA
| | - Hengameh Shams
- Departments of Bioengineering and Mechanical Engineering, University of California, Berkeley, CA 94720, USA
| | - Frank V Cochran
- Departments of Bioengineering and Chemical Engineering, Stanford University, Stanford, CA 94305, USA
| | - José L Alonso
- Leukocyte Biology and Inflammation Program, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA 02129, USA; Structural Biology Program, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA 02129, USA; Division of Nephrology/Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA 02129, USA
| | - James R Kintzing
- Departments of Bioengineering and Chemical Engineering, Stanford University, Stanford, CA 94305, USA
| | - Kiavash Garakani
- Departments of Bioengineering and Mechanical Engineering, University of California, Berkeley, CA 94720, USA
| | - Brian D Adair
- Leukocyte Biology and Inflammation Program, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA 02129, USA; Structural Biology Program, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA 02129, USA; Division of Nephrology/Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA 02129, USA
| | - Jian-Ping Xiong
- Leukocyte Biology and Inflammation Program, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA 02129, USA; Structural Biology Program, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA 02129, USA; Division of Nephrology/Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA 02129, USA
| | - Mohammad R K Mofrad
- Departments of Bioengineering and Mechanical Engineering, University of California, Berkeley, CA 94720, USA
| | - Jennifer R Cochran
- Departments of Bioengineering and Chemical Engineering, Stanford University, Stanford, CA 94305, USA
| | - M Amin Arnaout
- Leukocyte Biology and Inflammation Program, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA 02129, USA; Structural Biology Program, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA 02129, USA; Division of Nephrology/Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA 02129, USA.
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17
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Abstract
Phagocytosis refers to the active process that allows cells to take up large particulate material upon binding to surface receptors. The discovery of phagocytosis in 1883 by Elie Metchnikoff, leading to the concept that specialized cells are implicated in the defense against microbes, was one of the starting points of the field of immunology. After more than a century of research, phagocytosis is now appreciated to be a widely used process that enables the cellular uptake of a remarkable variety of particles, including bacteria, fungi, parasites, viruses, dead cells, and assorted debris and solid materials. Uptake of foreign particles is performed almost exclusively by specialized myeloid cells, commonly termed "professional phagocytes": neutrophils, monocytes, macrophages, and dendritic cells. Phagocytosis of microbes not only stops or at least restricts the spread of infection but also plays an important role in regulating the innate and adaptive immune responses. Activation of the myeloid cells upon phagocytosis leads to the secretion of cytokines and chemokines that convey signals to a variety of immune cells. Moreover, foreign antigens generated by the degradation of microbes following phagocytosis are loaded onto the major histocompatibility complex for presentation to specific T lymphocytes. However, phagocytosis is not restricted to professional myeloid phagocytes; an expanding diversity of cell types appear capable of engulfing apoptotic bodies and debris, playing a critical role in tissue remodeling and in the clearance of billions of effete cells every day.
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18
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Phagocytosis: A Fundamental Process in Immunity. BIOMED RESEARCH INTERNATIONAL 2017; 2017:9042851. [PMID: 28691037 PMCID: PMC5485277 DOI: 10.1155/2017/9042851] [Citation(s) in RCA: 313] [Impact Index Per Article: 39.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 02/07/2017] [Accepted: 04/18/2017] [Indexed: 01/12/2023]
Abstract
One hundred years have passed since the death of Élie Metchnikoff (1845-1916). He was the first to observe the uptake of particles by cells and realized the importance of this process for the host response to injury and infection. He also was a strong advocate of the role of phagocytosis in cellular immunity, and with this he gave us the basis for our modern understanding of inflammation and the innate and acquired immune responses. Phagocytosis is an elegant but complex process for the ingestion and elimination of pathogens, but it is also important for the elimination of apoptotic cells and hence fundamental for tissue homeostasis. Phagocytosis can be divided into four main steps: (i) recognition of the target particle, (ii) signaling to activate the internalization machinery, (iii) phagosome formation, and (iv) phagolysosome maturation. In recent years, the use of new tools of molecular biology and microscopy has provided new insights into the cellular mechanisms of phagocytosis. In this review, we present a general view of our current knowledge on phagocytosis. We emphasize novel molecular findings, particularly on phagosome formation and maturation, and discuss aspects that remain incompletely understood.
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19
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Monti M, Iommelli F, De Rosa V, Carriero MV, Miceli R, Camerlingo R, Di Minno G, Del Vecchio S. Integrin-dependent cell adhesion to neutrophil extracellular traps through engagement of fibronectin in neutrophil-like cells. PLoS One 2017; 12:e0171362. [PMID: 28166238 PMCID: PMC5293257 DOI: 10.1371/journal.pone.0171362] [Citation(s) in RCA: 30] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/21/2016] [Accepted: 01/18/2017] [Indexed: 12/22/2022] Open
Abstract
Neutrophil extracellular traps (NETs), originally recognized as a host defense mechanism, were reported to promote thrombosis and metastatic dissemination of cancer cells. Here we tested the role of integrins α5β1 and ανβ3 in the adhesion of cancer cells to NETs. Neutrophil-like cells stimulated with calcium ionophore (A23187) were used as a stable source of cell-free NETs-enriched suspensions. Using NETs as an adhesion substrate, two human K562 cell lines, differentially expressing α5β1 and ανβ3 integrins, were subjected to adhesion assays in the presence or absence of DNAse 1, blocking antibodies against α5β1 or ανβ3, alone or in combination with DNAse 1, and Proteinase K. As expected DNAse 1 treatment strongly inhibited adhesion of both cell lines to NETs. An equivalent significant reduction of cell adhesion to NETs was obtained after treatment of cells with blocking antibodies against α5β1 or ανβ3 indicating that both integrins were able to mediate cell adhesion to NETs. Furthermore, the combination of DNAse 1 and anti-integrin antibody treatment almost completely blocked cell adhesion. Western blot analysis and immunoprecipitation experiments showed a dose-dependent increase of fibronectin levels in samples from stimulated neutrophil-like cells and a direct or indirect interaction of fibronectin with histone H3. Finally, co-immunolocalization studies with confocal microscopy showed that fibronectin and citrullinated histone H3 co-localize inside the web-structure of NETs. In conclusion, our study showed that α5β1 and ανβ3 integrins mediate cell adhesion to NETs by binding to their common substrate fibronectin. Therefore, in addition to mechanical trapping and aspecific adsorption of different cell types driven by DNA/histone complexes, NETs may provide specific binding sites for integrin-mediated cell adhesion of neutrophils, platelets, endothelial and cancer cells thus promoting intimate interactions among these cells.
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Affiliation(s)
- Marcello Monti
- Dipartimento di Medicina Clinica e Chirurgia, Università degli Studi di Napoli “Federico II”, Naples, Italy
| | - Francesca Iommelli
- Istituto di Biostrutture e Bioimmagini, Consiglio Nazionale delle Ricerche, Naples, Italy
| | - Viviana De Rosa
- Istituto di Biostrutture e Bioimmagini, Consiglio Nazionale delle Ricerche, Naples, Italy
| | - Maria Vincenza Carriero
- Dipartimento di Oncologia Sperimentale, IRCCS Istituto Nazionale Tumori “Fondazione G. Pascale”, Naples, Italy
| | - Roberta Miceli
- Dipartimento di Oncologia Sperimentale, IRCCS Istituto Nazionale Tumori “Fondazione G. Pascale”, Naples, Italy
| | - Rosa Camerlingo
- Dipartimento di Oncologia Sperimentale, IRCCS Istituto Nazionale Tumori “Fondazione G. Pascale”, Naples, Italy
| | - Giovanni Di Minno
- Dipartimento di Medicina Clinica e Chirurgia, Università degli Studi di Napoli “Federico II”, Naples, Italy
| | - Silvana Del Vecchio
- Istituto di Biostrutture e Bioimmagini, Consiglio Nazionale delle Ricerche, Naples, Italy
- Dipartimento di Scienze Biomediche Avanzate, Università degli Studi di Napoli “Federico II”, Naples, Italy
- * E-mail:
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20
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Johnson CM, O'Brien XM, Byrd AS, Parisi VE, Loosely AJ, Li W, Witt H, Faridi MH, LeFort CT, Gupta V, Kim M, Reichner JS. Integrin Cross-Talk Regulates the Human Neutrophil Response to Fungal β-Glucan in the Context of the Extracellular Matrix: A Prominent Role for VLA3 in the Antifungal Response. THE JOURNAL OF IMMUNOLOGY 2016; 198:318-334. [PMID: 27852744 DOI: 10.4049/jimmunol.1502381] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/09/2015] [Accepted: 10/20/2016] [Indexed: 11/19/2022]
Abstract
Candida albicans infection produces elongated hyphae resistant to phagocytic clearance compelling alternative neutrophil effector mechanisms to destroy these physically large microbial structures. Additionally, all tissue-based neutrophilic responses to fungal infections necessitate contact with the extracellular matrix (ECM). Neutrophils undergo a rapid, ECM-dependent mechanism of homotypic aggregation and NETosis in response to C. albicans mediated by the β2 integrin, complement receptor 3 (CR3, CD11b/CD18, αMβ2). Neither homotypic aggregation nor NETosis occurs when human neutrophils are exposed either to immobilized fungal β-glucan or to C. albicans hyphae without ECM. The current study provides a mechanistic basis to explain how matrix controls the antifungal effector functions of neutrophils under conditions that preclude phagocytosis. We show that CR3 ligation initiates a complex mechanism of integrin cross-talk resulting in differential regulation of the β1 integrins VLA3 (α3β1) and VLA5 (α5β1). These β1 integrins control distinct antifungal effector functions in response to either fungal β-glucan or C. albicans hyphae and fibronectin, with VLA3 inducing homotypic aggregation and VLA5 regulating NETosis. These integrin-dependent effector functions are controlled temporally whereby VLA5 and CR3 induce rapid, focal NETosis early after binding fibronectin and β-glucan. Within minutes, CR3 undergoes inside-out auto-activation that drives the downregulation of VLA5 and the upregulation of VLA3 to support neutrophil swarming and aggregation. Forcing VLA5 to remain in the activated state permits NETosis but prevents homotypic aggregation. Therefore, CR3 serves as a master regulator during the antifungal neutrophil response, controlling the affinity states of two different β1 integrins, which in turn elicit distinct effector functions.
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Affiliation(s)
- Courtney M Johnson
- Division of Surgical Research, Department of Surgery, Rhode Island Hospital, Providence, RI 02903.,Warren Alpert Medical School, Brown University, Providence, RI 02912.,Graduate Program in Pathobiology, Brown University, Providence, RI 02912
| | - Xian M O'Brien
- Division of Surgical Research, Department of Surgery, Rhode Island Hospital, Providence, RI 02903.,Warren Alpert Medical School, Brown University, Providence, RI 02912
| | - Angel S Byrd
- Division of Surgical Research, Department of Surgery, Rhode Island Hospital, Providence, RI 02903.,Warren Alpert Medical School, Brown University, Providence, RI 02912.,Graduate Program in Pathobiology, Brown University, Providence, RI 02912
| | - Valentina E Parisi
- Division of Surgical Research, Department of Surgery, Rhode Island Hospital, Providence, RI 02903.,Graduate Program in Pathobiology, Brown University, Providence, RI 02912
| | - Alex J Loosely
- Department of Physics, Brown University, Providence, RI 02912
| | - Wei Li
- Division of Surgical Research, Department of Surgery, Rhode Island Hospital, Providence, RI 02903
| | - Hadley Witt
- Division of Surgical Research, Department of Surgery, Rhode Island Hospital, Providence, RI 02903.,Warren Alpert Medical School, Brown University, Providence, RI 02912.,Graduate Program in Pathobiology, Brown University, Providence, RI 02912
| | - Mohd H Faridi
- Department of Internal Medicine, Rush University Medical Center, Chicago, IL 60612
| | - Craig T LeFort
- Division of Surgical Research, Department of Surgery, Rhode Island Hospital, Providence, RI 02903.,Warren Alpert Medical School, Brown University, Providence, RI 02912
| | - Vineet Gupta
- Department of Internal Medicine, Rush University Medical Center, Chicago, IL 60612
| | - Minsoo Kim
- David H. Smith Center for Vaccine Biology and Immunology, Department of Microbiology and Immunology, University of Rochester, Rochester, NY 14642
| | - Jonathan S Reichner
- Division of Surgical Research, Department of Surgery, Rhode Island Hospital, Providence, RI 02903.,Warren Alpert Medical School, Brown University, Providence, RI 02912.,Graduate Program in Pathobiology, Brown University, Providence, RI 02912
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21
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O'Brien XM, Reichner JS. Neutrophil Integrins and Matrix Ligands and NET Release. Front Immunol 2016; 7:363. [PMID: 27698655 PMCID: PMC5027203 DOI: 10.3389/fimmu.2016.00363] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/29/2016] [Accepted: 09/02/2016] [Indexed: 12/23/2022] Open
Abstract
Neutrophils are motile and responsive to tissue injury and infection. As neutrophils emigrate from the bloodstream and migrate toward a site of affliction, they encounter the tissue extracellular matrix (ECM) and thereby engage integrins. Our laboratory studies the neutrophilic response to the fungal pathogen Candida albicans either in the filamentous state of the microbe or to the purified pathogen-associated molecular pattern, β-glucan. We have gained an appreciation for the role of integrins in regulating the neutrophil anti-Candida response and how the presence or absence of ECM can drive experimental outcome. The β2 integrin CR3 (complement receptor 3; αMβ2; Mac-1; CD11b/CD18) plays an important role in fungal recognition by its ability to bind β-glucan at a unique lectin-like domain. The presence of ECM differentially regulates essential neutrophil anti-fungal functions, including chemotaxis, respiratory burst, homotypic aggregation, and the release of neutrophil extracellular traps (NETs). We have shown that NET release to C. albicans hyphae or immobilized β-glucan occurs rapidly and without the requirement for respiratory burst on ECM. This is in contrast to the more frequently reported mechanisms of NETosis to other pathogens without the context of ECM, which occur after a prolonged lag period and require respiratory burst. As expected for an ECM-dependent phenotype, NETosis and other neutrophil functions are dependent on specific integrins. The focus of this review is the role of ECM ligation by neutrophil integrins as it pertains to host defense functions with an emphasis on lessons we have learned studying the anti-Candida response of human neutrophils.
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Affiliation(s)
- Xian M O'Brien
- Division of Surgical Research, Department of Surgery, Rhode Island Hospital, Providence, RI, USA; Warren Alpert Medical School, Brown University, Providence, RI, USA
| | - Jonathan S Reichner
- Division of Surgical Research, Department of Surgery, Rhode Island Hospital, Providence, RI, USA; Warren Alpert Medical School, Brown University, Providence, RI, USA
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22
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Di Pietro P, Zaccaro L, Comegna D, Del Gatto A, Saviano M, Snyders R, Cossement D, Satriano C, Rizzarelli E. Silver nanoparticles functionalized with a fluorescent cyclic RGD peptide: a versatile integrin targeting platform for cells and bacteria. RSC Adv 2016. [DOI: 10.1039/c6ra21568h] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
A green fluorescent RGD peptide–silver nanoparticle platform to target integrin receptors in cells and bacterial studies.
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Affiliation(s)
- P. Di Pietro
- Department of Chemical Sciences
- University of Catania
- Consorzio Interuniversitario di Ricerca in Chimica dei Metalli nei Sistemi Biologici (C.I.R.C.M.S.B.)
- 95125 Catania
- Italy
| | - L. Zaccaro
- Institute of Biostructure and Bioimaging (IBB) of the Italian National Research Council (CNR)
- Napoli
- Italy
| | - D. Comegna
- Institute of Biostructure and Bioimaging (IBB) of the Italian National Research Council (CNR)
- Napoli
- Italy
| | - A. Del Gatto
- Institute of Biostructure and Bioimaging (IBB) of the Italian National Research Council (CNR)
- Napoli
- Italy
| | - M. Saviano
- Institute of Crystallography (IC) of the Italian National Research Council (CNR)
- Bari
- Italy
| | - R. Snyders
- Chimie des Interactions Plasma Surface (ChIPS)
- Research Institute for Materials Science and Engineering
- Université de Mons (UMONS)
- Belgium
- Materia Nova Research Center
| | | | - C. Satriano
- Department of Chemical Sciences
- University of Catania
- Consorzio Interuniversitario di Ricerca in Chimica dei Metalli nei Sistemi Biologici (C.I.R.C.M.S.B.)
- 95125 Catania
- Italy
| | - Enrico Rizzarelli
- Department of Chemical Sciences
- University of Catania
- Consorzio Interuniversitario di Ricerca in Chimica dei Metalli nei Sistemi Biologici (C.I.R.C.M.S.B.)
- 95125 Catania
- Italy
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23
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Dissociation of HSV gL from gH by αvβ6- or αvβ8-integrin promotes gH activation and virus entry. Proc Natl Acad Sci U S A 2015; 112:E3901-10. [PMID: 26157134 DOI: 10.1073/pnas.1506846112] [Citation(s) in RCA: 45] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/28/2023] Open
Abstract
Herpes simplex virus (HSV) is an important human pathogen. It enters cells through an orchestrated process that requires four essential glycoproteins, gD, gH/gL, and gB, activated in cascade fashion by receptor-binding and signaling. gH/gL heterodimer is conserved across the Herpesviridae family. HSV entry is enabled by gH/gL interaction with αvβ6- or αvβ8-integrin receptors. We report that the interaction of virion gH/gL with integrins resulted in gL dissociation and its release in the medium. gL dissociation occurred if all components of the entry apparatus-receptor-bound gD and gB-were present and was prevented if entry was blocked by a neutralizing monoclonal antibody to gH or by a mutation in gH. We propose that (i) gL dissociation from gH/gL is part of the activation of HSV glycoproteins, critical for HSV entry; and (ii) gL is a functional inhibitor of gH and maintains gH in an inhibited form until receptor-bound gD and integrins signal to gH/gL.
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24
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Reyes R, Monjas A, Yánez-Mó M, Cardeñes B, Morlino G, Gilsanz A, Machado-Pineda Y, Lafuente E, Monk P, Sánchez-Madrid F, Cabañas C. Different states of integrin LFA-1 aggregation are controlled through its association with tetraspanin CD9. BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH 2015; 1853:2464-80. [PMID: 26003300 DOI: 10.1016/j.bbamcr.2015.05.018] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/11/2014] [Revised: 05/11/2015] [Accepted: 05/14/2015] [Indexed: 12/19/2022]
Abstract
The tetraspanin CD9 has been shown to interact with different members of the β1 and β3 subfamilies of integrins, regulating through these interactions cell adhesion, migration and signaling. Based on confocal microscopy co-localization and on co-immunoprecipitation results, we report here that CD9 associates with the β2 integrin LFA-1 in different types of leukocytes including T, B and monocytic cells. This association is resistant to stringent solubilization conditions which, together with data from chemical crosslinking, in situ Proximity Ligation Assays and pull-down experiments, suggest a primary/direct type of interaction mediated by the Large Extracellular Loop of the tetraspanin. CD9 exerts inhibitory effects on the adhesive function of LFA-1 and on LFA-1-dependent leukocyte cytotoxic activity. The mechanism responsible for this negative regulation exerted by CD9 on LFA-1 adhesion does not involve changes in the affinity state of this integrin but seems to be related to alterations in its state of aggregation.
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Affiliation(s)
- Raquel Reyes
- Centro de Biología Molecular Severo Ochoa (CSIC-UAM), 28049 Madrid, Spain; Departamento de Biología, Facultad de Ciencias, Universidad Autónoma de Madrid, 28049 Madrid, Spain
| | - Alicia Monjas
- Centro de Biología Molecular Severo Ochoa (CSIC-UAM), 28049 Madrid, Spain
| | - María Yánez-Mó
- Unidad de Investigación, Hospital Santa Cristina, Instituto de Investigación Sanitaria La Princesa (IIS-IP), 28006 Madrid, Spain; Departamento de Biología Molecular, Facultad de Ciencias, Universidad Autonoma de Madrid, 28049 Madrid, Spain
| | - Beatriz Cardeñes
- Centro de Biología Molecular Severo Ochoa (CSIC-UAM), 28049 Madrid, Spain
| | - Giulia Morlino
- Departamento de Biología Vascular e Inflamación, Centro Nacional de Investigaciones Cardiovasculares (CNIC), 28029 Madrid, Spain
| | - Alvaro Gilsanz
- Centro de Biología Molecular Severo Ochoa (CSIC-UAM), 28049 Madrid, Spain
| | | | - Esther Lafuente
- Departamento de Microbiología I, Area de Inmunología, Facultad de Medicina, Universidad Complutense de Madrid, 28040 Madrid, Spain
| | - Peter Monk
- University of Sheffield Medical School, Sheffield S10 2RX, UK
| | - Francisco Sánchez-Madrid
- Departamento de Biología Vascular e Inflamación, Centro Nacional de Investigaciones Cardiovasculares (CNIC), 28029 Madrid, Spain; Servicio de Inmunología, Hospital de la Princesa, Instituto de Investigación Sanitaria La Princesa (IIS-IP), 28006 Madrid, Spain
| | - Carlos Cabañas
- Centro de Biología Molecular Severo Ochoa (CSIC-UAM), 28049 Madrid, Spain; Departamento de Microbiología I, Area de Inmunología, Facultad de Medicina, Universidad Complutense de Madrid, 28040 Madrid, Spain.
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25
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Fujita M, Zhu K, Fujita CK, Zhao M, Lam KS, Kurth MJ, Takada YK, Takada Y. Proinflammatory secreted phospholipase A2 type IIA (sPLA-IIA) induces integrin activation through direct binding to a newly identified binding site (site 2) in integrins αvβ3, α4β1, and α5β1. J Biol Chem 2015; 290:259-71. [PMID: 25398877 PMCID: PMC4281730 DOI: 10.1074/jbc.m114.579946] [Citation(s) in RCA: 34] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/08/2014] [Revised: 11/12/2014] [Indexed: 11/06/2022] Open
Abstract
Integrins are activated by signaling from inside the cell (inside-out signaling) through global conformational changes of integrins. We recently discovered that fractalkine activates integrins in the absence of CX3CR1 through the direct binding of fractalkine to a ligand-binding site in the integrin headpiece (site 2) that is distinct from the classical RGD-binding site (site 1). We propose that fractalkine binding to the newly identified site 2 induces activation of site 1 though conformational changes (in an allosteric mechanism). We reasoned that site 2-mediated activation of integrins is not limited to fractalkine. Human secreted phospholipase A2 type IIA (sPLA2-IIA), a proinflammatory protein, binds to integrins αvβ3 and α4β1 (site 1), and this interaction initiates a signaling pathway that leads to cell proliferation and inflammation. Human sPLA2-IIA does not bind to M-type receptor very well. Here we describe that sPLA2-IIA directly activated purified soluble integrin αvβ3 and transmembrane αvβ3 on the cell surface. This activation did not require catalytic activity or M-type receptor. Docking simulation predicted that sPLA2-IIA binds to site 2 in the closed-headpiece of αvβ3. A peptide from site 2 of integrin β1 specifically bound to sPLA2-IIA and suppressed sPLA2-IIA-induced integrin activation. This suggests that sPLA2-IIA activates αvβ3 through binding to site 2. sPLA2-IIA also activated integrins α4β1 and α5β1 in a site 2-mediated manner. We recently identified small compounds that bind to sPLA2-IIA and suppress integrin-sPLA2-IIA interaction (e.g. compound 21 (Cmpd21)). Cmpd21 effectively suppressed sPLA2-IIA-induced integrin activation. These results define a novel mechanism of proinflammatory action of sPLA2-IIA through integrin activation.
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Affiliation(s)
- Masaaki Fujita
- From the Departments of Dermatology and Biochemistry and Molecular Medicine, UC Davis School of Medicine, Sacramento, California 95817, Department of Clinical Immunology and Rheumatology, The Tazuke-Kofukai Medical Research Institute, Kitano Hospital, Osaka 530-8480, Japan
| | - Kan Zhu
- From the Departments of Dermatology and
| | - Chitose K Fujita
- Department of Clinical Immunology and Rheumatology, The Tazuke-Kofukai Medical Research Institute, Kitano Hospital, Osaka 530-8480, Japan
| | - Min Zhao
- From the Departments of Dermatology and
| | - Kit S Lam
- Biochemistry and Molecular Medicine, UC Davis School of Medicine, Sacramento, California 95817
| | - Mark J Kurth
- Department of Chemistry, UC Davis, Davis, California 95616, and
| | - Yoko K Takada
- From the Departments of Dermatology and Biochemistry and Molecular Medicine, UC Davis School of Medicine, Sacramento, California 95817
| | - Yoshikazu Takada
- From the Departments of Dermatology and Biochemistry and Molecular Medicine, UC Davis School of Medicine, Sacramento, California 95817,
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26
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Fujita M, Takada YK, Takada Y. The chemokine fractalkine can activate integrins without CX3CR1 through direct binding to a ligand-binding site distinct from the classical RGD-binding site. PLoS One 2014; 9:e96372. [PMID: 24789099 PMCID: PMC4008574 DOI: 10.1371/journal.pone.0096372] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/13/2013] [Accepted: 04/07/2014] [Indexed: 11/19/2022] Open
Abstract
The chemokine domain of fractalkine (FKN-CD) binds to the classical RGD-binding site of αvβ3 and that the resulting ternary complex formation (integrin-FKN-CX3CR1) is critical for CX3CR1 signaling and FKN-induced integrin activation. However, only certain cell types express CX3CR1. Here we studied if FKN-CD can activate integrins in the absence of CX3CR1. We describe that WT FKN-CD activated recombinant soluble αvβ3 in cell-free conditions, but the integrin-binding defective mutant of FKN-CD (K36E/R37E) did not. This suggests that FKN-CD can activate αvβ3 in the absence of CX3CR1 through the direct binding of FKN-CD to αvβ3. WT FKN-CD activated αvβ3 on CX3CR1-negative cells (K562 and CHO) but K36E/R37E did not, suggesting that FKN-CD can activate integrin at the cellular levels in a manner similar to that in cell-free conditions. We hypothesized that FKN-CD enhances ligand binding to the classical RGD-binding site (site 1) through binding to a second binding site (site 2) that is distinct from site 1 in αvβ3. To identify the possible second FKN-CD binding site we performed docking simulation of αvβ3-FKN-CD interaction using αvβ3 with a closed inactive conformation as a target. The simulation predicted a potential FKN-CD-binding site in inactive αvβ3 (site 2), which is located at a crevice between αv and β3 on the opposite side of site 1 in the αvβ3 headpiece. We studied if FKN-CD really binds to site 2 using a peptide that is predicted to interact with FKN-CD in site 2. Notably the peptide specifically bound to FKN-CD and effectively suppressed integrin activation by FKN-CD. This suggests that FKN-CD actually binds to site 2, and this leads to integrin activation. We obtained very similar results in α4β1 and α5β1. The FKN binding to site 2 and resulting integrin activation may be a novel mechanism of integrin activation and of FKN signaling.
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Affiliation(s)
- Masaaki Fujita
- Department of Dermatology, Biochemistry and Molecular Medicine, UC Davis School of Medicine, Sacramento, California, United States of America
| | - Yoko K. Takada
- Department of Dermatology, Biochemistry and Molecular Medicine, UC Davis School of Medicine, Sacramento, California, United States of America
| | - Yoshikazu Takada
- Department of Dermatology, Biochemistry and Molecular Medicine, UC Davis School of Medicine, Sacramento, California, United States of America
- * E-mail:
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27
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Gianni T, Salvioli S, Chesnokova LS, Hutt-Fletcher LM, Campadelli-Fiume G. αvβ6- and αvβ8-integrins serve as interchangeable receptors for HSV gH/gL to promote endocytosis and activation of membrane fusion. PLoS Pathog 2013; 9:e1003806. [PMID: 24367260 PMCID: PMC3868510 DOI: 10.1371/journal.ppat.1003806] [Citation(s) in RCA: 80] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/10/2013] [Accepted: 10/15/2013] [Indexed: 01/20/2023] Open
Abstract
Herpes simplex virus (HSV) - and herpesviruses in general - encode for a multipartite entry/fusion apparatus. In HSV it consists of the HSV-specific glycoprotein D (gD), and three additional glycoproteins, gH/gL and gB, conserved across the Herpesviridae family and responsible for the execution of fusion. According to the current model, upon receptor binding, gD propagates the activation to gH/gL and to gB in a cascade fashion. Questions remain about how the cascade of activation is controlled and how it is synchronized with virion endocytosis, to avoid premature activation and exhaustion of the glycoproteins. We considered the possibility that such control might be carried out by as yet unknown receptors. Indeed, receptors for HSV gB, but not for gH/gL, have been described. In other members of the Herpesviridae family, such as Epstein-Barr virus, integrin receptors bind gH/gL and trigger conformational changes in the glycoproteins. We report that αvβ6- and αvβ8-integrins serve as receptors for HSV entry into experimental models of keratinocytes and other epithelial and neuronal cells. Evidence rests on loss of function experiments, in which integrins were blocked by antibodies or silenced, and gain of function experiments in which αvβ6-integrin was expressed in integrin-negative cells. αvβ6- and αvβ8-integrins acted independently and are thus interchangeable. Both bind gH/gL with high affinity. The interaction profoundly affects the route of HSV entry and directs the virus to acidic endosomes. In the case of αvβ8, but not αvβ6-integrin, the portal of entry is located at lipid microdomains and requires dynamin 2. Thus, a major role of αvβ6- or αvβ8-integrin in HSV infection appears to be to function as gH/gL receptors and to promote virus endocytosis. We propose that placing the gH/gL activation under the integrin trigger point enables HSV to synchronize virion endocytosis with the cascade of glycoprotein activation that culminates in execution of fusion. In order to infect their hosts and cause disease, viruses must enter their host cells. The human pathogen herpes simplex virus (HSV) - and herpesviruses in general - are equipped with a complex, multipartite entry apparatus, made of four glycoproteins – gD, gH/gL, gB. These glycoproteins must be activated in a timely, coordinated manner. According to the current model, the flux of activation goes from receptor-bound gD, to gH/gL and gB. The premature activation, and hence exhaustion of the glycoproteins must also be prevented. We report on a checkpoint at the gH/gL level. Specifically, αvβ6- and αvβ8-integrins serve as receptors for HSV entry into keratinocytes and other epithelial and neuronal cells. Both bind gH/gL with high affinity. The interaction profoundly affects the pathway of HSV entry, promoting HSV endocytosis into acidic endosomes. For αvβ8-integrin, the portal of entry is at lipid microdomains and requires dynamin 2. We propose that, by placing the activation of gH/gL under control of an integrin trigger point, HSV can synchronize virion endocytosis with the cascade of activation that culminates in the execution of fusion between the virion envelope and cellular membranes.
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Affiliation(s)
- Tatiana Gianni
- Department of Experimental, Diagnostic and Specialty Medicine, Alma Mater Studiorum–University of Bologna, Bologna, Italy
| | - Stefano Salvioli
- Department of Experimental, Diagnostic and Specialty Medicine, Alma Mater Studiorum–University of Bologna, Bologna, Italy
| | - Liudmila S. Chesnokova
- Department of Microbiology and Immunology, Center for Molecular and Tumor Virology and Feist-Weiller Cancer Center, Louisiana State University Health Sciences Center, Shreveport, Louisiana, United States of America
| | - Lindsey M. Hutt-Fletcher
- Department of Microbiology and Immunology, Center for Molecular and Tumor Virology and Feist-Weiller Cancer Center, Louisiana State University Health Sciences Center, Shreveport, Louisiana, United States of America
| | - Gabriella Campadelli-Fiume
- Department of Experimental, Diagnostic and Specialty Medicine, Alma Mater Studiorum–University of Bologna, Bologna, Italy
- * E-mail:
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28
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Das M, Ithychanda S, Qin J, Plow EF. Mechanisms of talin-dependent integrin signaling and crosstalk. BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES 2013; 1838:579-88. [PMID: 23891718 DOI: 10.1016/j.bbamem.2013.07.017] [Citation(s) in RCA: 92] [Impact Index Per Article: 7.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/26/2013] [Revised: 07/03/2013] [Accepted: 07/15/2013] [Indexed: 01/01/2023]
Abstract
Cells undergo dynamic remodeling of the cytoskeleton during adhesion and migration on various extracellular matrix (ECM) substrates in response to physiological and pathological cues. The major mediators of such cellular responses are the heterodimeric adhesion receptors, the integrins. Extracellular or intracellular signals emanating from different signaling cascades cause inside-out signaling of integrins via talin, a cystokeletal protein that links integrins to the actin cytoskeleton. Various integrin subfamilies communicate with each other and growth factor receptors under diverse cellular contexts to facilitate or inhibit various integrin-mediated functions. Since talin is an essential mediator of integrin activation, much of the integrin crosstalk would therefore be influenced by talin. However, despite the existence of an extensive body of knowledge on the role of talin in integrin activation and as a stabilizer of ECM-actin linkage, information on its role in regulating inter-integrin communication is limited. This review will focus on the structure of talin, its regulation of integrin activation and discuss its potential role in integrin crosstalk. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters. Guest Editor: Jean Claude Hervé.
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Affiliation(s)
- Mitali Das
- Department of Molecular Cardiology, Joseph J. Jacobs Center for Thrombosis and Vascular Biology, Lerner Research Institute, Cleveland Clinic
| | - Sujay Ithychanda
- Department of Molecular Cardiology, Joseph J. Jacobs Center for Thrombosis and Vascular Biology, Lerner Research Institute, Cleveland Clinic
| | - Jun Qin
- Department of Molecular Cardiology, Joseph J. Jacobs Center for Thrombosis and Vascular Biology, Lerner Research Institute, Cleveland Clinic
| | - Edward F Plow
- Department of Molecular Cardiology, Joseph J. Jacobs Center for Thrombosis and Vascular Biology, Lerner Research Institute, Cleveland Clinic
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29
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Hussain N, Siegel A, Ge Y, Jordan R, Naumann C. Bilayer asymmetry influences integrin sequestering in raft-mimicking lipid mixtures. Biophys J 2013; 104:2212-21. [PMID: 23708361 PMCID: PMC3660629 DOI: 10.1016/j.bpj.2013.04.020] [Citation(s) in RCA: 39] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/27/2012] [Revised: 03/15/2013] [Accepted: 04/09/2013] [Indexed: 12/25/2022] Open
Abstract
There is growing recognition that lipid heterogeneities in cellular membranes play an important role in the distribution and functionality of membrane proteins. However, the detection and characterization of such heterogeneities at the cellular level remains challenging. Here we report on the poorly understood relationship between lipid bilayer asymmetry and membrane protein sequestering in raft-mimicking model membrane mixtures using a powerful experimental platform comprised of confocal spectroscopy XY-scan and photon-counting histogram analyses. This experimental approach is utilized to probe the domain-specific sequestering and oligomerization state of αvβ3 and α5β1 integrins in bilayers, which contain coexisting liquid-disordered/liquid-ordered (ld/lo) phase regions exclusively in the top leaflet of the bilayer (bottom leaflet contains ld phase). Comparison with previously reported integrin sequestering data in bilayer-spanning lo-ld phase separations demonstrates that bilayer asymmetry has a profound influence on αvβ3 and α5β1 sequestering behavior. For example, both integrins sequester preferentially to the lo phase in asymmetric bilayers, but to the ld phase in their symmetric counterparts. Furthermore, our data show that bilayer asymmetry significantly influences the role of native ligands in integrin sequestering.
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Affiliation(s)
- Noor F. Hussain
- Department of Chemistry and Chemical Biology, Indiana University-Purdue University Indianapolis, Indiana
| | - Amanda P. Siegel
- Department of Chemistry and Chemical Biology, Indiana University-Purdue University Indianapolis, Indiana
| | - Yifan Ge
- Department of Chemistry and Chemical Biology, Indiana University-Purdue University Indianapolis, Indiana
| | - Rainer Jordan
- Makromolekulare Chemie, Dresden University of Technology, Dresden, Germany
| | - Christoph A. Naumann
- Department of Chemistry and Chemical Biology, Indiana University-Purdue University Indianapolis, Indiana
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30
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de Morrée A, Flix B, Bagaric I, Wang J, van den Boogaard M, Grand Moursel L, Frants RR, Illa I, Gallardo E, Toes R, van der Maarel SM. Dysferlin regulates cell adhesion in human monocytes. J Biol Chem 2013; 288:14147-14157. [PMID: 23558685 DOI: 10.1074/jbc.m112.448589] [Citation(s) in RCA: 44] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Dysferlin is mutated in a group of muscular dystrophies commonly referred to as dysferlinopathies. It is highly expressed in skeletal muscle, where it is important for sarcolemmal maintenance. Recent studies show that dysferlin is also expressed in monocytes. Moreover, muscle of dysferlinopathy patients is characterized by massive immune cell infiltrates, and dysferlin-negative monocytes were shown to be more aggressive and phagocytose more particles. This suggests that dysferlin deregulation in monocytes might contribute to disease progression, but the molecular mechanism is unclear. Here we show that dysferlin expression is increased with differentiation in human monocytes and the THP1 monocyte cell model. Freshly isolated monocytes of dysferlinopathy patients show deregulated expression of fibronectin and fibronectin-binding integrins, which is recapitulated by transient knockdown of dysferlin in THP1 cells. Dysferlin forms a protein complex with these integrins at the cell membrane, and its depletion impairs cell adhesion. Moreover, patient macrophages show altered adhesion and motility. These findings suggest that dysferlin is involved in regulating cellular interactions and provide new insight into dysferlin function in inflammatory cells.
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Affiliation(s)
- Antoine de Morrée
- Department of Human Genetics, Leiden University Medical Center, 2333 ZA Leiden, The Netherlands
| | - Bàrbara Flix
- Servei de Neurologia, Laboratori de Neurologia Experimental, Hospital de la Santa Creu i Sant Pau i Institut de Recerca de HSCSP, Universitat Autònoma de Barcelona and Centro de Investigación Biomédica en Red de Enfermedades Neurodegenerativas (CIBERNED), 08193 Bellaterra, Spain
| | - Ivana Bagaric
- Department of Human Genetics, Leiden University Medical Center, 2333 ZA Leiden, The Netherlands
| | - Jun Wang
- Department of Rheumatology, Leiden University Medical Center, 2333 ZA Leiden, The Netherlands
| | | | - Laure Grand Moursel
- Department of Human Genetics, Leiden University Medical Center, 2333 ZA Leiden, The Netherlands
| | - Rune R Frants
- Department of Human Genetics, Leiden University Medical Center, 2333 ZA Leiden, The Netherlands
| | - Isabel Illa
- Servei de Neurologia, Laboratori de Neurologia Experimental, Hospital de la Santa Creu i Sant Pau i Institut de Recerca de HSCSP, Universitat Autònoma de Barcelona and Centro de Investigación Biomédica en Red de Enfermedades Neurodegenerativas (CIBERNED), 08193 Bellaterra, Spain
| | - Eduard Gallardo
- Servei de Neurologia, Laboratori de Neurologia Experimental, Hospital de la Santa Creu i Sant Pau i Institut de Recerca de HSCSP, Universitat Autònoma de Barcelona and Centro de Investigación Biomédica en Red de Enfermedades Neurodegenerativas (CIBERNED), 08193 Bellaterra, Spain
| | - Rene Toes
- Department of Rheumatology, Leiden University Medical Center, 2333 ZA Leiden, The Netherlands
| | - Silvère M van der Maarel
- Department of Human Genetics, Leiden University Medical Center, 2333 ZA Leiden, The Netherlands.
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31
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Moore SJ, Leung CL, Norton HK, Cochran JR. Engineering agatoxin, a cystine-knot peptide from spider venom, as a molecular probe for in vivo tumor imaging. PLoS One 2013; 8:e60498. [PMID: 23573262 PMCID: PMC3616073 DOI: 10.1371/journal.pone.0060498] [Citation(s) in RCA: 38] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/10/2012] [Accepted: 02/26/2013] [Indexed: 11/18/2022] Open
Abstract
BACKGROUND Cystine-knot miniproteins, also known as knottins, have shown great potential as molecular scaffolds for the development of targeted therapeutics and diagnostic agents. For this purpose, previous protein engineering efforts have focused on knottins based on the Ecballium elaterium trypsin inhibitor (EETI) from squash seeds, the Agouti-related protein (AgRP) neuropeptide from mammals, or the Kalata B1 uterotonic peptide from plants. Here, we demonstrate that Agatoxin (AgTx), an ion channel inhibitor found in spider venom, can be used as a molecular scaffold to engineer knottins that bind with high-affinity to a tumor-associated integrin receptor. METHODOLOGY/PRINCIPAL FINDINGS We used a rational loop-grafting approach to engineer AgTx variants that bound to αvβ3 integrin with affinities in the low nM range. We showed that a disulfide-constrained loop from AgRP, a structurally-related knottin, can be substituted into AgTx to confer its high affinity binding properties. In parallel, we identified amino acid mutations required for efficient in vitro folding of engineered integrin-binding AgTx variants. Molecular imaging was used to evaluate in vivo tumor targeting and biodistribution of an engineered AgTx knottin compared to integrin-binding knottins based on AgRP and EETI. Knottin peptides were chemically synthesized and conjugated to a near-infrared fluorescent dye. Integrin-binding AgTx, AgRP, and EETI knottins all generated high tumor imaging contrast in U87MG glioblastoma xenograft models. Interestingly, EETI-based knottins generated significantly lower non-specific kidney imaging signals compared to AgTx and AgRP-based knottins. CONCLUSIONS/SIGNIFICANCE In this study, we demonstrate that AgTx, a knottin from spider venom, can be engineered to bind with high affinity to a tumor-associated receptor target. This work validates AgTx as a viable molecular scaffold for protein engineering, and further demonstrates the promise of using tumor-targeting knottins as probes for in vivo molecular imaging.
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Affiliation(s)
- Sarah J. Moore
- Department of Bioengineering, Stanford University, Stanford, California, United States of America
| | - Cheuk Lun Leung
- Department of Chemical Engineering, Stanford University, Stanford, California, United States of America
| | - Heidi K. Norton
- Department of Bioengineering, Stanford University, Stanford, California, United States of America
| | - Jennifer R. Cochran
- Department of Bioengineering, Stanford University, Stanford, California, United States of America
- Department of Chemical Engineering, Stanford University, Stanford, California, United States of America
- Stanford Cancer Institute and Bio-X Program, Stanford, California, United States of America
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Gilk SD, Cockrell DC, Luterbach C, Hansen B, Knodler LA, Ibarra JA, Steele-Mortimer O, Heinzen RA. Bacterial colonization of host cells in the absence of cholesterol. PLoS Pathog 2013; 9:e1003107. [PMID: 23358892 PMCID: PMC3554619 DOI: 10.1371/journal.ppat.1003107] [Citation(s) in RCA: 50] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/25/2012] [Accepted: 11/16/2012] [Indexed: 12/21/2022] Open
Abstract
Reports implicating important roles for cholesterol and cholesterol-rich lipid rafts in host-pathogen interactions have largely employed sterol sequestering agents and biosynthesis inhibitors. Because the pleiotropic effects of these compounds can complicate experimental interpretation, we developed a new model system to investigate cholesterol requirements in pathogen infection utilizing DHCR24−/− mouse embryonic fibroblasts (MEFs). DHCR24−/− MEFs lack the Δ24 sterol reductase required for the final enzymatic step in cholesterol biosynthesis, and consequently accumulate desmosterol into cellular membranes. Defective lipid raft function by DHCR24−/− MEFs adapted to growth in cholesterol-free medium was confirmed by showing deficient uptake of cholera-toxin B and impaired signaling by epidermal growth factor. Infection in the absence of cholesterol was then investigated for three intracellular bacterial pathogens: Coxiella burnetii, Salmonella enterica serovar Typhimurium, and Chlamydia trachomatis. Invasion by S. Typhimurium and C. trachomatis was unaltered in DHCR24−/− MEFs. In contrast, C. burnetii entry was significantly decreased in −cholesterol MEFs, and also in +cholesterol MEFs when lipid raft-associated αVβ3 integrin was blocked, suggesting a role for lipid rafts in C. burnetii uptake. Once internalized, all three pathogens established their respective vacuolar niches and replicated normally. However, the C. burnetii-occupied vacuole within DHCR24−/− MEFs lacked the CD63-postive material and multilamellar membranes typical of vacuoles formed in wild type cells, indicating cholesterol functions in trafficking of multivesicular bodies to the pathogen vacuole. These data demonstrate that cholesterol is not essential for invasion and intracellular replication by S. Typhimurium and C. trachomatis, but plays a role in C. burnetii-host cell interactions. Clustered receptors associated with cholesterol-rich microdomains, termed lipid rafts, are thought to provide plasma membrane signaling platforms that bacterial pathogens can subvert to gain entry into host cells. Moreover, cholesterol has been implicated as a critical structural lipid of several pathogen-occupied vacuoles. Cumulative data supporting these models have principally been derived using inhibitors of cholesterol metabolism and various sterol sequestering compounds, agents that can lack specificity and cause unwanted cellular affects. Here, we employed a new system to investigate pathogen reliance on cholesterol for host cell colonization that utilizes mouse embryonic fibroblasts that can synthesize precursor sterols, but not cholesterol. Cells lacking cholesterol displayed strong defects in lipid raft-based signaling. However, no defects were observed in entry, vacuole development, and growth of Salmonella enterica and Chlamydia trachomatis, bacterial pathogens previously shown to rely on cholesterol for optimal host cell parasitism. Entry by Coxiella burnetii, the bacterial cause of human Q fever, was significantly decreased in cholesterol-negative cells as was trafficking of membranous material to the pathogen vacuole. However, subsequent bacterial replication was unaltered. Our results should prompt a reevaluation of the overall importance of cholesterol in bacterial pathogenesis with the described experimental system providing an alternative approach for such studies.
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Affiliation(s)
- Stacey D. Gilk
- Coxiella Pathogenesis Section, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, United States of America
| | - Diane C. Cockrell
- Coxiella Pathogenesis Section, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, United States of America
| | - Courtney Luterbach
- Salmonella-Host Cell Interactions Section, Laboratory of Intracellular Parasites, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, United States of America
| | - Bryan Hansen
- Microscopy Unit, Research Technology Branch, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, United States of America
| | - Leigh A. Knodler
- Salmonella-Host Cell Interactions Section, Laboratory of Intracellular Parasites, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, United States of America
| | - J. Antonio Ibarra
- Salmonella-Host Cell Interactions Section, Laboratory of Intracellular Parasites, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, United States of America
| | - Olivia Steele-Mortimer
- Salmonella-Host Cell Interactions Section, Laboratory of Intracellular Parasites, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, United States of America
| | - Robert A. Heinzen
- Coxiella Pathogenesis Section, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, United States of America
- * E-mail:
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Integrins and small GTPases as modulators of phagocytosis. INTERNATIONAL REVIEW OF CELL AND MOLECULAR BIOLOGY 2013; 302:321-54. [PMID: 23351714 DOI: 10.1016/b978-0-12-407699-0.00006-6] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Phagocytosis is the mechanism whereby cells engulf large particles. This process has long been recognized as a critical component of the innate immune response, which constitutes the organism's defense against microorganisms. In addition, phagocytic internalization of apoptotic cells or cell fragments plays important roles in tissue homeostasis and remodeling. Phagocytosis requires target interactions with receptors on the plasma membrane of the phagocytic cell. Integrins have been identified as important mediators of particle clearance, in addition to their well-established roles in cell adhesion, migration and mechanotransduction. Indeed, these ubiquitously expressed proteins impart phagocytic capacity to epithelial, endothelial and mesenchymal cell types. The importance of integrins in particle internalization is emphasized by the ability of microbial and viral pathogens to exploit their signaling pathways to invade host cells, and by the wide variety of disorders that arise from abnormalities in integrin-dependent phagocytic uptake.
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αvβ3-integrin is a major sensor and activator of innate immunity to herpes simplex virus-1. Proc Natl Acad Sci U S A 2012; 109:19792-7. [PMID: 23150579 DOI: 10.1073/pnas.1212597109] [Citation(s) in RCA: 51] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
Pathogens are sensed by Toll-like receptors (TLRs) and a growing number of non-TLR receptors. Integrins constitute a family of signaling receptors exploited by viruses and bacteria to access cells. By gain- and loss-of-function approaches we found that αvβ3-integrin is a sensor of and plays a crucial role in the innate defense against herpes simplex virus (HSV). αvβ3-integrin signaled through two pathways. One concurred with TLR2, affected activation/induction of interferons type 1 (IFNs-1), NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells), and a polarized set of cytokines and receptors. The virion glycoproteins gH/gL sufficed to induce IFN1 and NF-κB via this pathway. The other pathway was TLR2-independent, involved sarcoma (SRC)-spleen tyrosine kinase (SYK)-Caspase recruitment domain-containing protein 9 (CARD9)-TRIF (TIR-domain-containing adapter-inducing interferon-β), and affected interferon regulatory factor 3 and 7 (IRF3-IRF7). The importance of αvβ3-integrin-mediated defense is reflected in the observation that HSV evolved the immediate-early infected cellular protein 0 (ICP0) protein to counteract it. We propose that αvβ3-integrin is considered a class of non-TLR pattern recognition receptors, a role likely exerted toward viruses and bacteria that interact with integrins and mount an innate response.
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Fujita M, Takada YK, Takada Y. Integrins αvβ3 and α4β1 act as coreceptors for fractalkine, and the integrin-binding defective mutant of fractalkine is an antagonist of CX3CR1. THE JOURNAL OF IMMUNOLOGY 2012; 189:5809-19. [PMID: 23125415 DOI: 10.4049/jimmunol.1200889] [Citation(s) in RCA: 49] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
Abstract
The membrane-bound chemokine fractalkine (FKN, CX3CL1) on endothelial cells plays a role in leukocyte trafficking. The chemokine domain (FKN-CD) is sufficient for inducing FKN signaling (e.g., integrin activation), and FKN-CD binds to its receptor CX3CR1 on leukocytes. Whereas previous studies suggest that FKN-CD does not directly bind to integrins, our docking simulation studies predicted that FKN-CD directly interacts with integrin α(v)β(3). Consistent with this prediction, we demonstrated that FKN-CD directly bound to α(v)β(3) and α(4)β(1) at a very high affinity (K(D) of 3.0 × 10(-10) M to α(v)β(3) in 1 mM Mn(2+)). Also, membrane-bound FKN bound to integrins α(v)β(3) and α(4)β(1), suggesting that the FKN-CD/integrin interaction is biologically relevant. The binding site for FKN-CD in α(v)β(3) was similar to those for other known α(v)β(3) ligands. Wild-type FKN-CD induced coprecipitation of integrins and CX3CR1 in U937 cells, suggesting that FKN-CD induces ternary complex formation (CX3CR1, FKN-CD, and integrin). Based on the docking model, we generated an integrin-binding defective FKN-CD mutant (the K36E/R37E mutant). K36E/R37E was defective in ternary complex formation and integrin activation, whereas K36E/R37E still bound to CX3CR1. These results suggest that FKN-CD binding to CX3CR1 is not sufficient for FKN signaling, and that FKN-CD binding to integrins as coreceptors and the resulting ternary complex formation are required for FKN signaling. Notably, excess K36E/R37E suppressed integrin activation induced by wild-type FKN-CD and effectively suppressed leukocyte infiltration in thioglycollate-induced peritonitis. These findings suggest that K36E/R37E acts as a dominant-negative CX3CR1 antagonist and that FKN-CD/integrin interaction is a novel therapeutic target in inflammatory diseases.
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Affiliation(s)
- Masaaki Fujita
- Department of Dermatology, University of California Davis School of Medicine, Sacramento, CA 95817, USA
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Patton SM, Coe CL, Lubach GR, Connor JR. Quantitative proteomic analyses of cerebrospinal fluid using iTRAQ in a primate model of iron deficiency anemia. Dev Neurosci 2012; 34:354-65. [PMID: 23018452 DOI: 10.1159/000341919] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/24/2012] [Accepted: 07/16/2012] [Indexed: 12/17/2022] Open
Abstract
Iron deficiency affects nearly 2 billion people worldwide, with pregnant women and young children being most severely impacted. Sustained anemia during the first year of life can cause cognitive, attention and motor deficits, which may persist despite iron supplementation. We conducted iTRAQ analyses on cerebrospinal fluid (CSF) from infant monkeys (Macaca mulatta) to identify differential protein expression associated with early iron deficiency. CSF was collected from 5 iron-sufficient and 8 iron-deficient anemic monkeys at weaning age (6-7 months) and again at 12-14 months. Despite consumption of iron-fortified food after weaning, which restored hematological indices into the normal range, expression of 5 proteins in the CSF remained altered. Most of the proteins identified are involved in neurite outgrowth, migration or synapse formation. The results reveal novel ways in which iron deficiency undermines brain growth and results in aberrant neuronal migration and connections. Taken together with gene expression data from rodent models of iron deficiency, we conclude that significant alterations in neuroconnectivity occur in the iron-deficient brain, which may persist even after resolution of the hematological anemia. The compromised brain infrastructure could account for observations of behavioral deficits in children during and after the period of anemia.
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37
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Christophi GP, Gruber RC, Panos M, Christophi RL, Jubelt B, Massa PT. Interleukin-33 upregulation in peripheral leukocytes and CNS of multiple sclerosis patients. Clin Immunol 2012; 142:308-19. [PMID: 22189043 PMCID: PMC3288946 DOI: 10.1016/j.clim.2011.11.007] [Citation(s) in RCA: 82] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/22/2011] [Revised: 11/11/2011] [Accepted: 11/21/2011] [Indexed: 01/16/2023]
Abstract
Multiple sclerosis (MS) is an immune-mediated demyelinating disease of the central nervous system (CNS). Here we document for the first time that the cytokine IL-33 is upregulated in both the periphery and the CNS of MS patients. Plasma IL-33 was elevated in MS patients compared to normal subjects and a three-month treatment of MS patients with interferon β-1a resulted in a significant decrease of IL-33 levels. Similarly, stimulated cultured lymphocytes and macrophages from MS patients had elevated IL-33 levels compared to normal subjects. In parallel, the transcription factor NF-κB that mediates IL-33 transcription was also elevated in leukocytes of MS patients. IL-33 was elevated in normal-appearing white matter and plaque areas from MS brains and astrocytes were identified as an important source of IL-33 expression in the CNS. In summary, IL-33 levels are elevated in the periphery and CNS of MS patients, implicating IL-33 in the pathogenesis of MS.
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Affiliation(s)
- George P. Christophi
- Department of Microbiology & Immunology, Albert Einstein College of Medicine, Bronx, NY, USA
- Department of Medicine, Washington University School of Medicine, St. Louis, MO, USA
| | - Ross C. Gruber
- Department of Neurology, Albert Einstein College of Medicine, Bronx, NY, USA
- Department of Pathology, Albert Einstein College of Medicine, Bronx, NY, USA
| | - Michael Panos
- Department of Neurology, Albert Einstein College of Medicine, Bronx, NY, USA
| | - Rebecca L. Christophi
- Department of Microbiology & Immunology, Albert Einstein College of Medicine, Bronx, NY, USA
| | - Burk Jubelt
- Department of Neurology, Albert Einstein College of Medicine, Bronx, NY, USA
- Department of Microbiology & Immunology, Albert Einstein College of Medicine, Bronx, NY, USA
| | - Paul T. Massa
- Department of Neurology, Albert Einstein College of Medicine, Bronx, NY, USA
- Department of Microbiology & Immunology, Albert Einstein College of Medicine, Bronx, NY, USA
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Flannagan RS, Jaumouillé V, Grinstein S. The Cell Biology of Phagocytosis. ANNUAL REVIEW OF PATHOLOGY-MECHANISMS OF DISEASE 2012; 7:61-98. [PMID: 21910624 DOI: 10.1146/annurev-pathol-011811-132445] [Citation(s) in RCA: 677] [Impact Index Per Article: 52.1] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Affiliation(s)
- Ronald S. Flannagan
- Program in Cell Biology, Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada;
| | - Valentin Jaumouillé
- Program in Cell Biology, Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada;
| | - Sergio Grinstein
- Program in Cell Biology, Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada;
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Glycoproteins D of equine herpesvirus type 1 (EHV-1) and EHV-4 determine cellular tropism independently of integrins. J Virol 2011; 86:2031-44. [PMID: 22171258 DOI: 10.1128/jvi.06555-11] [Citation(s) in RCA: 37] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Equine herpesvirus type 1 (EHV-1) and EHV-4 are genetically and antigenically very similar, but their pathogenic potentials are strikingly different. The differences in pathogenicity between both viruses seem to be reflected in cellular host range: EHV-1 can readily be propagated in many cell types of multiple species, while EHV-4 entry and replication appear to be restricted mainly to equine cells. The clear difference in cellular tropism may well be associated with differences in the gene products involved in virus entry and/or spread from cell to cell. Here we show that (i) most of the EHV-1 permissive cell lines became resistant to EHV-1 expressing EHV-4 glycoprotein D (gD4) and the opposite was observed for EHV-4 harboring EHV-1 gD (gD1). (ii) The absence of integrins did not inhibit entry into and replication of EHV-1 in CHO-K1 or peripheral blood mononuclear cells (PBMC). Furthermore, integrin-negative K562 cells did not acquire the ability to bind to gD1 when αVβ3 integrin was overexpressed. (iii) PBMC could be infected with similar efficiencies by both EHV-1 and EHV-4 in vitro. (iv) In contrast to results for equine fibroblasts and cells of endothelial or epithelial origin, we were unable to block entry of EHV-1 or EHV-4 into PBMC with antibodies directed against major histocompatibility complex class I (MHC-I), a result that indicates that these viruses utilize a different receptor(s) to infect PBMC. Cumulatively, we provide evidence that efficient EHV-1 and EHV-4 entry is dependent mainly on gD, which can bind to multiple cell surface receptors, and that gD has a defining role with respect to cellular host range of EHV-1 and EHV-4.
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40
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Siegel A, Kimble-Hill A, Garg S, Jordan R, Naumann C. Native ligands change integrin sequestering but not oligomerization in raft-mimicking lipid mixtures. Biophys J 2011; 101:1642-50. [PMID: 21961590 PMCID: PMC3183796 DOI: 10.1016/j.bpj.2011.08.040] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/10/2011] [Revised: 08/17/2011] [Accepted: 08/30/2011] [Indexed: 11/16/2022] Open
Abstract
Distinct lipid environments, including lipid rafts, are increasingly recognized as a crucial factor affecting membrane protein function in plasma membranes. Unfortunately, an understanding of their role in membrane protein activation and oligomerization has remained elusive due to the challenge of characterizing these often small and transient plasma membrane heterogeneities in live cells. To address this difficulty, we present an experimental model membrane platform based on polymer-supported lipid bilayers containing stable raft-mimicking domains (type I) and homogeneous cholesterol-lipid mixtures (type II) into which transmembrane proteins are incorporated (α(v)β(3) and α(5)β(1) integrins). These flexible lipid platforms enable the use of confocal fluorescence spectroscopy, including the photon counting histogram method, in tandem with epifluorescence microscopy to quantitatively probe the effect of the binding of native ligands from the extracellular matrix ligands (vitronectin and fibronectin for α(v)β(3) and α(5)β(1), respectively) on domain-specific protein sequestration and on protein oligomerization state. We found that both α(v)β(3) and α(5)β(1) sequester preferentially to nonraft domains in the absence of extracellular matrix ligands, but upon ligand addition, α(v)β(3) sequesters strongly into raft-like domains and α(5)β(1) loses preference for either raft-like or nonraft-like domains. A corresponding photon counting histogram analysis showed that integrins exist predominantly in a monomeric state. No change was detected in oligomerization state upon ligand binding in either type I or type II bilayers, but a moderate increase in oligomerization state was observed for increasing concentrations of cholesterol. The combined findings suggest a mechanism in which changes in integrin sequestering are caused by ligand-induced changes in integrin conformation and/or dynamics that affect integrin-lipid interactions without altering the integrin oligomerization state.
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Affiliation(s)
- Amanda P. Siegel
- Department of Chemistry and Chemical Biology, Indiana University Purdue University, Indianapolis, Indiana
| | - Ann Kimble-Hill
- Department of Chemistry and Chemical Biology, Indiana University Purdue University, Indianapolis, Indiana
| | - Sumit Garg
- Department of Chemistry and Chemical Biology, Indiana University Purdue University, Indianapolis, Indiana
| | - Rainer Jordan
- Department Chemie, Technische Universität Dresden, Dresden, Germany
| | - Christoph A. Naumann
- Department of Chemistry and Chemical Biology, Indiana University Purdue University, Indianapolis, Indiana
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Geiger B, Yamada KM. Molecular architecture and function of matrix adhesions. Cold Spring Harb Perspect Biol 2011; 3:cshperspect.a005033. [PMID: 21441590 DOI: 10.1101/cshperspect.a005033] [Citation(s) in RCA: 391] [Impact Index Per Article: 27.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Cell adhesions mediate important bidirectional interactions between cells and the extracellular matrix. They provide an interactive interface between the extracellular chemical and physical environment and the cellular scaffolding and signaling machinery. This dynamic, reciprocal regulation of intracellular processes and the matrix is mediated by membrane receptors such as the integrins, as well as many other components that comprise the adhesome. Adhesome constituents assemble themselves into different types of cell adhesion structures that vary in molecular complexity and change over time. These cell adhesions play crucial roles in cell migration, proliferation, and determination of cell fate.
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Affiliation(s)
- Benjamin Geiger
- Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel.
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Yubero N, Jiménez-Marín A, Barbancho M, Garrido JJ. Two cDNAs coding for the porcine CD51 (αv) integrin subunit: cloning, expression analysis, adhesion assays and chromosomal localization. Gene 2011; 481:29-40. [PMID: 21549183 DOI: 10.1016/j.gene.2011.04.006] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/10/2011] [Revised: 04/11/2011] [Accepted: 04/15/2011] [Indexed: 02/06/2023]
Abstract
CD51 (α(v)) is an integrin chain that associates with multiple β integrin chains to form different receptor complexes that mediate important human processes. Pigs show substantial physiological, immunological and anatomical similarities to humans, and are therefore a good model system to study immunological and pathological processes. Here we report the cloning and characterization of two cDNAs produced by alternative splicing that encode two different porcine CD51 proteins that differ in five amino acid residues. Pig CD51 cDNAs encode polypeptides of 1046 or 1041 amino acid residues, respectively, that share with other mammalian homologous proteins a high percentage amino acid identity and the functional domains. Expression analysis of CD51 was carried out at two different levels. RT-PCR analysis revealed that both CD51 transcripts were expressed ubiquitously but heterogeneously, with the exception of some platelets in which only the smallest CD51 transcript was detected. A specific monoclonal antibody against a pig CD51 recombinant protein was made and used in the immunohistochemical localization of CD51 proteins. It showed that CD51 was mainly expressed in hematopoietic cells of myeloid linage, epithelial and endothelial cells, osteoclasts, nervous fibers and smooth muscle. Adhesion assays showed that in the presence of Mn(++) pig α(v)-CHO-B2 transfected cells increased their attachment to fibronectin and vitonectin, but not to fibrinogen. Finally, we localized the CD51 gene on the porcine chromosome 15 (SSC15), q23-q26.
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Affiliation(s)
- Noemí Yubero
- Unidad de Genómica y Mejora Animal, Departamento de Genética, Universidad de Córdoba, Campus de Rabanales, 14071 Córdoba, Spain
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Hassan GS, Merhi Y, Mourad W. CD40 ligand: a neo-inflammatory molecule in vascular diseases. Immunobiology 2011; 217:521-32. [PMID: 21529993 DOI: 10.1016/j.imbio.2011.03.010] [Citation(s) in RCA: 60] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/24/2011] [Accepted: 03/29/2011] [Indexed: 11/17/2022]
Abstract
CD40 Ligand (CD40L), a member of the TNF family, was initially thought to be solely implicated in thymus-dependent humoral responses. However, work by several groups showed that CD40L plays a more global role in various systems. Recent evidence has outlined an important role for CD40L in the physiopathology of the vascular system. Indeed, by interacting with its principal receptor, CD40, or with the recently identified receptors, namely αIIbβ3, α5β1, and Mac-1 integrins, CD40L displayed many biological functions in different types of vascular cells. In addition, the CD40L system was demonstrated a major player in the pathology of vascular diseases, such as atherosclerosis and restenosis. This review outlines the expression pattern and the functional properties of CD40L and its receptors at different cellular levels in the vascular system. In addition, we thoroughly describe evidence showing the implication of CD40L interactions in atherosclerosis, restenosis, and their associated clinical complications.
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Affiliation(s)
- Ghada S Hassan
- Laboratoire d'Immunologie Cellulaire et Moléculaire, Centre Hospitalier de l'Université de Montréal, Hôpital Saint-Luc, Montréal QC H2X 1P1, Canada
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Okada Y, Nishikawa JI, Semma M, Ichikawa A. Induction of integrin β3 in PGE2-stimulated adhesion of mastocytoma P-815 cells to the Arg-Gly-Asp-enriched fragment of fibronectin. Biochem Pharmacol 2011; 81:866-72. [DOI: 10.1016/j.bcp.2011.01.010] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/01/2010] [Revised: 01/13/2011] [Accepted: 01/14/2011] [Indexed: 12/19/2022]
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45
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Kimura RH, Jones DS, Jiang L, Miao Z, Cheng Z, Cochran JR. Functional mutation of multiple solvent-exposed loops in the Ecballium elaterium trypsin inhibitor-II cystine knot miniprotein. PLoS One 2011; 6:e16112. [PMID: 21364742 PMCID: PMC3041754 DOI: 10.1371/journal.pone.0016112] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/19/2010] [Accepted: 12/08/2010] [Indexed: 01/04/2023] Open
Abstract
BACKGROUND The Ecballium elaterium trypsin inhibitor (EETI-II), a 28-amino acid member of the knottin family of peptides, contains three interwoven disulfide bonds that form multiple solvent-exposed loops. Previously, the trypsin binding loop of EETI-II has been engineered to confer binding to several alternative molecular targets. Here, EETI-II was further explored as a molecular scaffold for polypeptide engineering by evaluating the ability to mutate two of its structurally adjacent loops. METHODOLOGY/PRINCIPAL FINDINGS Yeast surface display was used to engineer an EETI-II mutant containing two separate integrin binding epitopes. The resulting knottin peptide was comprised of 38 amino acids, and contained 11- and 10-residue loops compared to wild-type EETI-II, which naturally contains 6- and 5-residue loops, respectively. This knottin peptide bound to α(v)β(3) and α(v)β(5) integrins with affinities in the low nanomolar range, but bound weakly to the related integrins α(5)β(1) and α(iib)β(3). In addition, the engineered knottin peptide inhibited tumor cell adhesion to vitronectin, an extracellular matrix protein that binds to α(v)β(3) and α(v)β(5) integrins. A (64)Cu radiolabeled version of this knottin peptide demonstrated moderate serum stability and excellent tumor-to-muscle and tumor-to-blood ratios by positron emission tomography imaging in human tumor xenograft models. Tumor uptake was ∼3-5% injected dose per gram (%ID/g) at one hour post injection, with rapid clearance of probe through the kidneys. CONCLUSIONS/SIGNIFICANCE We demonstrated that multiple loops of EETI-II can be mutated to bind with high affinity to tumor-associated integrin receptors. The resulting knottin peptide contained 21 (>50%) non-native amino acids within two mutated loops, indicating that extended loop lengths and sequence diversity were well tolerated within the EETI-II scaffold. A radiolabeled version of this knottin peptide showed promise for non-invasive imaging of integrin expression in living subjects. However, reduced serum and metabolic stability were observed compared to an engineered integrin-binding EETI-II knottin peptide containing only one mutated loop.
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Affiliation(s)
- Richard H. Kimura
- Department of Radiology, Molecular Imaging Program at Stanford (MIPS), Cancer Center, Bio-X Program, Stanford University, Stanford, California, United States of America
| | - Douglas S. Jones
- Department of Bioengineering, Cancer Center, Bio-X Program, Stanford University, Stanford, California, United States of America
| | - Lei Jiang
- Department of Radiology, Molecular Imaging Program at Stanford (MIPS), Cancer Center, Bio-X Program, Stanford University, Stanford, California, United States of America
| | - Zheng Miao
- Department of Radiology, Molecular Imaging Program at Stanford (MIPS), Cancer Center, Bio-X Program, Stanford University, Stanford, California, United States of America
| | - Zhen Cheng
- Department of Radiology, Molecular Imaging Program at Stanford (MIPS), Cancer Center, Bio-X Program, Stanford University, Stanford, California, United States of America
| | - Jennifer R. Cochran
- Department of Bioengineering, Cancer Center, Bio-X Program, Stanford University, Stanford, California, United States of America
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{alpha}V{beta}3-integrin routes herpes simplex virus to an entry pathway dependent on cholesterol-rich lipid rafts and dynamin2. Proc Natl Acad Sci U S A 2010; 107:22260-5. [PMID: 21135248 DOI: 10.1073/pnas.1014923108] [Citation(s) in RCA: 73] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/02/2023] Open
Abstract
HSVs enter cells in a receptor-dependent [nectin1 or herpesviruses entry mediator (HVEM)] fashion by fusion of the viral envelope with plasma membrane (neutral pH compartment), by endocytosis into neutral or acidic compartments, or by macropinocytosis/phagocytosis. The cellular determinants of the route of entry are unknown. Here, we asked what cellular factors determine the pathway of HSV entry. CHO cells lack β(3)-integrin and the respective α-subunits' heterodimers. We report that, in the absence of α(V)β(3)-integrin, HSV enters CHO-nectin1 cells through a pathway independent of cholesterol-rich rafts and dynamin2. In the presence of α(V)β(3)-integrin, HSV enters CHO-nectin1 cells through a pathway dependent on cholesterol-rich rafts and dynamin2. HSV enters J-nectin1 and 293T cells through a neutral compartment independent of cholesterol-rich rafts and dynamin2. α(V)β(3)-integrin overexpression in these cells modifies the route of entry to an acidic compartment dependent on cholesterol-rich rafts and dynamin2, hence similar to that in α(V)β(3)-integrin-positive CHO-nectin1 cells. In some cells, the diversion of entry from an integrin- and raft-independent pathway to an acidic compartment requiring cholesterol-rich lipids rafts and dynamin2 is irreversible. Indeed, HSV cannot infect CHO-nectin1-α(V)β(3) cells through any compartment when the αvβ3-integrin-dependent pathway is blocked by anti-integrin antibody, anti-dynamin2, or anti-acidification drugs. We conclude that the αvβ3-integrin is a determinant in the choice of HSV entry pathway into cells. Because the pathway dictated by αvβ3-integrin is through lipid rafts, the platforms for a number of Toll-like receptors, current findings raise the possibility that αvβ3-integrin acts as a sentinel of innate immunity.
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Jaumouillé V, Grinstein S. Receptor mobility, the cytoskeleton, and particle binding during phagocytosis. Curr Opin Cell Biol 2010; 23:22-9. [PMID: 21074980 DOI: 10.1016/j.ceb.2010.10.006] [Citation(s) in RCA: 68] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/04/2010] [Revised: 10/11/2010] [Accepted: 10/17/2010] [Indexed: 02/04/2023]
Abstract
Particle engulfment during phagocytosis has long been appreciated to be an active, actin-driven process. By contrast, the preceding stage--securing the target to the surface of the phagocyte--was thought to result from the passive diffusion of receptors along the membrane towards their ligands on the particle surface. Recent evidence, however, challenges this notion, demonstrating that receptors do not diffuse freely along the phagocyte surface and that actin polymerization and tyrosine phosphorylation are required for optimal particle binding. The interpretation and significance of these observations are the subject of this opinion piece.
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Affiliation(s)
- Valentin Jaumouillé
- Cell Biology Program, Hospital for Sick Children, 555 University Ave., Toronto M5G 1X8, Canada
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Dykstra T, Utermoehlen O, Haas A. Defined particle ligands trigger specific defense mechanisms of macrophages. Innate Immun 2010; 17:388-402. [PMID: 20682584 DOI: 10.1177/1753425910374889] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022] Open
Abstract
Phagocytosis is a receptor-mediated process for sequestration and inactivation of infectious microbes. It can be triggered by microbial surface compounds or particle-attached host proteins. We monitored the effector functions of murine bone marrow-derived macrophages (BMMs) in response to polystyrene-streptavidin beads coated with the defined ligands IgG1, β-glucan, mannan, complement factors C1q or iC3b, or fibronectin (FN). Cell-autonomous effector mechanisms (uptake, phagosome maturation, cytokine responses and killing activity) were differentially triggered. All particle-ligand complexes stimulated the release of nitric oxide, but only beads coated with IgG, complement factors or FN caused production of superoxide. Beads coated with C1q, iC3b or FN strongly stimulated the secretion of pro-inflammatory TNF-α, IL-6, and IL-1β and also of anti-inflammatory IL-10. Escherichia coli coated with C1q, iC3b or FN was killed much less efficiently than with any of the other ligands, depending on the presence of IL-10 activity. This indicated an important role of IL-10 as regulator of cell-autonomous immune functions of macrophages. Our data show that defined ligands on microbial surfaces are interesting candidates to activate innate defense mechanisms selectively and specifically.
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Friedrichs J, Helenius J, Müller DJ. Stimulated single-cell force spectroscopy to quantify cell adhesion receptor crosstalk. Proteomics 2010; 10:1455-62. [PMID: 20127696 DOI: 10.1002/pmic.200900724] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
Abstract
To control their attachment to substrates and other cells, cells regulate their adhesion receptors. One regulatory process is receptor crosstalk, where the binding of one type of cell adhesion molecule influences the activity of another type. To identify such crosstalk and gain insight into their mechanisms, we developed the stimulated single-cell force spectroscopy assay. In this assay, the influence of a cells adhesion to one substrate on the strength of its adhesion to a second substrate is examined. The assay quantifies the adhesion of the cell and the contributions of specific adhesion receptors. This allows mechanisms by which the adhesion is regulated to be determined. Using the assay we identified crosstalk between collagen-binding integrin alpha(1)beta(1) and fibronectin-binding integrin alpha(5)beta(1) in HeLa cells. This crosstalk was unidirectional, from integrin alpha(1)beta(1) to integrin alpha(5)beta(1), and functioned by regulating the endocytosis of integrin alpha(5)beta(1). The single-cell assay should be expandable for the screening and quantification of crosstalk between various cell adhesion molecules and other cell surface receptors.
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Affiliation(s)
- Jens Friedrichs
- Biotechnology Center, University of Technology Dresden, Dresden, Germany
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Silverman AP, Kariolis MS, Cochran JR. Cystine-knot peptides engineered with specificities for αIIbβ3 or αIIbβ3 and αvβ3 integrins are potent inhibitors of platelet aggregation. J Mol Recognit 2010; 24:127-35. [DOI: 10.1002/jmr.1036] [Citation(s) in RCA: 36] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
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