Investigation of molecular mechanisms underlying the role of ICAM-3 in the phagocytic clearance of apoptotic leukocytes

Lead Research Organisation: Aston University
Department Name: Sch of Life and Health Sciences

Abstract

Damaged, infected, aged or unwanted cells in the body are induced to die via a process known as apoptosis. The final step in this process is the removal (phagocytosis) of dying cells by healthy neighbouring cells where the cell corpses are eaten and degraded in a safe and controlled environment. A range of molecules have been suggested to play a role in this process though the amount of information relating to molecules recognised on dying cells is limited. It has been suggested that changes at the surface of dying cells result in the exposure of signals or flags that identify the cell, to viable neighbours, as dying and ready for removal. Relatively little is currently known about the nature of such 'eat me' signals. Lipids at the cell surface are known to change during death with phosphatidylserine (PS) becoming exposed at the cell surface where in viable cells it is usually maintained within the inner leaflet of the cell membrane. Once exposed PS is recognised by receptors on viable cells and mediates dying cell clearance. A change in another molecule, ICAM-3 (Intercellular Adhesion Molecule-3) on the surface of dying leukocytes have been implicated in the recognition and removal of dying leukocytes by professional phagocytes, macrophages. ICAM-3 is a molecule found only on leukocytes and, when present on viable cells, is important in the initiation of immune responses by facilitating cell-cell interactions. To mediate this interaction ICAM-3 binds to counter-receptors including the integrin LFA-1 and the non-integrin DC-SIGN. Previous work has indicated that ICAM-3 on dying cells loses its ability to bind to its prototypic counter-receptor LFA-1. This apoptosis-related loss of function is associated with a gain of alternate function where ICAM-3 becomes able to bind to another, as yet unidentified, receptor - a function that is important in mediating the phagocytic clearance of apoptotic leukocytes. The removal of dying leukocytes is especially important for the resolution of inflammation and the control of immune responses. Failure to remove leukocytes at the appropriate time may lead to chronic inflammatory conditions and autoimmune disease. A better understanding of the molecules and processes involved within the clearance of dying leukocytes will be central to future strategies for improving human health and well-being. This project seeks to identify the changes that occur in ICAM-3 that underlie the loss of function (loss of LFA-1 binding) and the gain of function (ability to bind other receptor(s) and mediate clearance of dying leukocytes) that is reported during apoptosis. The change of function associated with ICAM-3 at the apoptotic cell surface may be due to changes in ICAM-3 location, partner molecules or structure (e.g. sugar content of ICAM-3) and any such changes may arise from or contribute to changes in the mobility of ICAM-3 as cells undergo apoptosis. Within this project we will identify and characterise changes in the structure and/or environment of ICAM-3 occurring during apoptosis and will relate these changes to dead cell clearance.

Technical Summary

Unwanted cells are removed in vivo through a carefully executed programme known as apoptosis that culminates in the safe and controlled removal of dying cells in an efficient manner. This application addresses the nature of molecular changes at the surface of dying cells that are fundamental to the success of apoptotic cell clearance. ICAM-3 (the most heavily glycosylated member of the Immunoglobulin Super Family) has previously been shown to change its function during apoptosis such that it loses its ability to bind LFA-1 (its viable cell counter-receptor) and gains the ability to bind a different receptor(s). This project seeks to identify the nature of the molecular changes underlying the role of ICAM-3 in apoptotic cell clearance. Using a range of fluorescence microscopy (including real-time analyses) and biochemical techniques we will analyse the localisation of ICAM-3 in relation to other cell surface molecules during apoptosis to identify whether a change of location and/or partner molecule(s) defines 'apoptotic cell-associated ICAM-3' and how this relates to phagocytic clearance. Real time studies will provide a powerful strategy for characterising the poorly characterised dynamic processes involved in apoptotic cell clearance. Further we will assess the structural integrity of ICAM-3 on viable and apoptotic cells to identify whether glycosylation changes underlie the ability of ICAM-3 to mediate apoptotic cell clearance. Finally we will characterise the ability of other IgSF members to function as ICAM-3 in clearance. This work will help better define the molecular processes and molecules involved in the clearance of apoptotic cells - a process central to the resolution of inflammation, control of immune responses, normal tissue homeostasis and development.
 
Description The aim of this programme of work was to assess the function of an important cell surface molecule of human white blood cells. This molecule, ICAM-3, changes function as cells die and it promotes removal of dying cells by viable cells. This process of dying cell removal (phagocytosis) is important in the control of inflammation. Our work has generated new reagents for the analysis of ICAM-3 (monoclonal antibodies: mAb). These are now commercially available. We identified how ICAM-3 is released from dying cells and how this release promote attraction of viable phagocytic cells. We have shown that we can modulate attraction of phagocytes to dying cells through the use of our mAb.
This programme of work progressed well and has:
(1) generated a novel anti-ICAM-3 blocking mAb from a panel of 17 anti-ICAM-3 mAbs;
(2) characterised apoptosis-associated changes in ICAM-3 distribution and shown these to be non-essential for its function in clearance;
(3) defined the mechanism for loss of ICAM-3 from the surface of cells during apoptosis;
(4) identified a role for released ICAM-3 in mediating phagocyte recruitment to sites of leukocyte apoptosis;
(5) formally defined ICAM-3 as a ligand for tethering apoptotic leukocytes to phagocytes; and
(6) partly characterised the receptor function necessary for the recognition of apoptotic ICAM-3, challenging the accepted wisdom that this incorporates a role for macrophage CD14.
Exploitation Route ICAM-3 mAbs have been produced and are now available throughout the world from commercial sources.

Our findings on the ability of ICAM-3 to modulate phagocyte recruitment to cell death has the potential for further analysis for therapeutic interventions in important diseases (e.g. cardiovascular disease).
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

URL http://www.nordicmubio.com/Products/ProductDetails/MUB2029P
 
Description The findings from this research programme have led to significant developments. The impact of these is still under development. Patents have been filed and the work is supporting new funding applications and a long-term programme of research addressing the importance of ICAM-3 and cell death in disease. The programme of work produced useful reagents that have been made available to the worldwide research community. The programme detailed the mechanism of ICAM-3 function. This has led to a number of publications (primary research, reviews, conference abstracts), has resulted in invited talks at international conference, has supported additional funding bids and a patent application that seeks to exploit the therapeutic potential of this programme of work. Ultimately therefore, the impact of this work will continue to develop. The reagents and technologies developed within this programme provide valuable opportunities for future research within the field of inflammatory disease with the potential to impact on disease therapy and quality of life. This is still early stage impact with the patent and publication arising in 2011. Patent applications are seeking approval worldwide
First Year Of Impact 2011
Sector Education,Healthcare,Manufacturing, including Industrial Biotechology
 
Description A novel therapy for lymphoma & atherosclerosis - identification of commercialisation approaches
Amount £10,717 (GBP)
Funding ID BB/S000011/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 05/2018 
End 11/2018
 
Description BBSRC Industrial Case Studentships
Amount £94,126 (GBP)
Funding ID BB/L016974/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 09/2014 
End 09/2018
 
Description BBSRC Responsive Mode
Amount £585,454 (GBP)
Funding ID BB/M006298/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 01/2015 
End 12/2018
 
Description Towards defining the functional surface of extracellular vesicles in dementia
Amount £70,341 (GBP)
Funding ID ARUK-EG2018A-006 
Organisation Alzheimer's Research UK 
Sector Charity/Non Profit
Country United Kingdom
Start 02/2018 
End 02/2019
 
Title Method to inhibit recruitment of monocytes and macrophages by an ICAM-3 inhibitor 
Description The present invention relates generally to methods and materials for modulating the recruitment of macrophages or monocytes to sites at which they may contribute to disease initiation or progression. Embodiments of the disclosure comprise providing a modulator of the activity of ICAM-3 or proximal to the site. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact This is a patent application where migration of phagocytes to dying cells may be reduced, for therapeutic gain, by reagents targeting ICAM-3. Such reagents may include our antibody. Further research is currently underway to assess the potential benefit of this within an in vivo situation. Thus this is a developing area. 
 
Title METHOD TO INHIBIT RECRUITMENT OF MONOCYTES AND MACROPHAGES BY AN ICAM-3 INHIBITOR 
Description The present disclosure relates generally to methods and materials for modulating the recruitment of macrophages or monocytes to sites at which they may contribute to disease initiation or progression. Embodiments of the disclosure comprise providing a modulator of the activity of ICAM- 3 or proximal to the site. 
IP Reference WO2012046001 
Protection Patent application published
Year Protection Granted 2012
Licensed No
Impact Work is ongoing.
 
Description Invited conference talk - Cambridge SelectBio (Extracellular Vesicles: Biology and Therapeutic Potential) - PI Prof Devitt 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact 2016 SelectBio: Extracellular Vesicles Biology & Therapeutic Potential (Cambridge). Invited talk.

This conference presentation at the SelectBio conference formed the basis of closer working relationships with other members of the scientific field of Extracellular Vesicles.
Invited conference talk - Rotterdam 2016 ISEV (International Society for Extracellular Vesicles) - PI Prof Devitt
Year(s) Of Engagement Activity 2016
URL https://selectbiosciences.com/conferences/biographies.aspx?speaker=1235882&conf=eve2016
 
Description BBSRC Immunology Working Group Member 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact The initial meetings are designed to help direct BBSRC future funding for Immunology research. I was invited to become a member based on my research profile which is supported by the research funding on these grants from the NC3R and BBSRC.
Year(s) Of Engagement Activity 2016,2017
 
Description I'm a scientist 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact This was an online chat with schools over a two week period. It focused on my research and enabled students to have live Q&A sessions with me. The breadth of the talk also expanded to cover other relevant subject material (e.g. immunology and infection)

As a result of this I was asked to support placement students from a local academy. I've supported students for each of the last two years.
Year(s) Of Engagement Activity 2013
 
Description IZON symposium 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation keynote/invited speaker
Geographic Reach International
Primary Audience Industry/Business
Results and Impact This was a talk regarding our work on in vitro models of innate immune function. The talk stimulated questions and discussion from audience members (within and without my research field). This also stimulated discussion on how to analyse particles (e.g. liposomes) that are relevant to this work.

PhD student from Cardiff has now visited my lab for training. This student is from a different scientific field to mine. Additionally, discussion are now underway with other symposium attendees for future collaboration.
Year(s) Of Engagement Activity 2014
URL http://www.izon.com/media/news/highlights-from-izon-science-research-symposium-in-oxford/
 
Description Invited Chair of a Discussion session at ISEV 2016 - PI Prof Devitt 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact This conference at the International Congress of the International Society for Extracellular Vesicles is the world-leading platform for scientific discussions and presentation relating to Extracellular Vesicle research. The invite for PI (Prof Devitt) came as a result of the previous work presented at ISEV in 2014.
Year(s) Of Engagement Activity 2016
URL http://www.isev.org/page/isev2016
 
Description Invited conference talk - Rotterdam 2016 ISEV (International Society for Extracellular Vesicles) - PI Prof Devitt 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact This conference presentation at the International Congress of the International Society for Extracellular Vesicles formed the basis of close working relationships with other members of the scientific field of Extracellular Vesicles. Our research group was singled out for special note in the Conference Summit up talk. Our PDRA won top prize for the Oral Talk. The conference enabled closer working relationships between international and UK national research groups. It cemented an application to host a Royal Society meeting.

I have also been invited back to speak again in 2017.
Year(s) Of Engagement Activity 2016
URL http://www.isev.org/page/isev2016
 
Description Invited conference talk - Rotterdam ISEV 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact This conference presentation at the International Congress of the International Society for Extracellular Vesicles formed the basis of close working relationships with other members of the scientific field of Extracellular Vesicles. It has led to applications for funds from the Royal Society to hold a meeting in 2017. It has also led to important collaborative contacts with other universities.

I have also been invited back to speak again in 2016.
Year(s) Of Engagement Activity 2014
 
Description Invited research presentation at UK EV symposium Oxford 2016 - Prof Andrew Devitt 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact This was an invited research presentation at the UK premier Extracellular Vesicle conference 2016. A valuable networking opportunity with data presented by Prof Andrew Devitt from multiple BBSRC-funded work.
Year(s) Of Engagement Activity 2016
URL http://www.ukev.org.uk/UKEV2016.html
 
Description Twitter (@ProfDevitt) 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact This is a Twitter account for all research from the Devitt research group at Aston. It reaches to students (UG/PG), business, public and practitioners.
Year(s) Of Engagement Activity 2014,2015,2016,2017
URL https://twitter.com/ProfDevitt