Role of Apoptotic Cell-Derived Icam-3 Promotes Both Macrophage Chemoattraction to and Tethering of Apoptotic Cells

Introduction

Phagocytosis describes the process by which apoptotic cells are eliminated. Failure to remove these cells has been linked to a variety of pathological scenarios. The study of apoptotic cell-derived ICAM-3 (a human leukocyte-restricted immunoglobulin superfamily member) microparticles and its relation to macrophage chemoattraction to and tethering of apoptotic cells gives insight on the precise mechanism by which macrophages identify apoptotic cells and target them for elimination. A variety of anti-ICAM-3 monoclonal antibodies, particularly the MA4 that blocks apoptotic cell clearance by macrophages and apoptotic human leukocytes that are deficient or normal for ICAM-3, were used to demonstrate that ICAM-3 promotes a domain 1-2 dependent tethering interaction with phagocytes such as macrophages (Duvall et al,1985). The study also illustrates that the apoptosis-associated decrease in ICAM-3 levels is as a result of the ICAM-3 microparticles release to attract macrophages to apoptotic cells. Results obtained from the study suggest that microparticles derived from apoptotic cell surfaces and which contain ICAM-3 promote and facilitate the chemoattraction of macrophages to sites containing apoptotic cells and leukocytes undergoing cell death. The results also show that ICAM-3 plays a significant role in the mediation of subsequent tethering of dead cells to macrophages. The defined purpose of ICAM-3 during apoptosis and phagocytosis, as well as the defect in chemotaxis observed in microparticles deficient of ICAM-3, have suggested that ICAM-3 may have a crucial role as an adhesion molecule as well as the involvement in chemotaxis to move towards apoptotic human leukocytes. The information in this study may be of great importance and a significant step in the search for a cure for cancer.

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Methodology

To maintain development, tissue maintenance and control of immune responses, phagocytes efficiently remove cells undergoing programmed cell death (apoptosis). Macrophages are the primary cells that remove apoptotic cells. Currently, the molecular mechanisms used by macrophages to recognize and get rid of apoptotic cells are still being researched although a variety of phagocytic receptors as well as some apoptotic cell-surface molecules have been explained. Phagocytosis involves a multistage process that includes recruitment of macrophages through chemotaxis before apoptotic cell recognition, tethering, and phagocytosis, all of which are accompanied by immunomodulatory signaling. The most defined change that takes place on the cell surface of apoptotic cells is the exposure of the aminophospholipid phosphatidylserine (PS) that oversees the elimination process.

Some less-defined changes indicate that sugars, PS-oxidation, and exposure of intracellular components such as annexin I am involved. Previous research has suggested that the immunoglobulin superfamily (IgSF) member intercellular-adhesion molecule (ICAM)-3 expressed on human leukocytes changes functionality during apoptosis to facilitate the elimination of apoptotic leukocytes through the suggested generation of neo-epitopes that mark them for removal. However, recent research of CD31, another IgSF, indicates that there are molecules present on viable cell surfaces that may work to provide inhibitory signals that are lost during apoptosis, which enables cellular interactions such as CD31-CD31 in trans to transform into functional phagocytic interactions. The research also suggests that the loss of the inhibitory signals and the gaining of neo-epitopes occurs on the cell surface of apoptotic surfaces and they work together to eliminate the removal of the cells. The nature and roleICAM-3 in the multistage cell clearance process have not been established.

Results

The purpose of this study is to show how apoptotic cell-associated ICAM-3 functions to tether apoptotic cells to phagocytes. During apoptosis, surface protein levels are reduced when part of the membrane is lost into microparticles. Such microparticles from apoptotic B cells display chemoattractive properties by relying on the chemokine fractalkine (CX3CL1) which marks the cell and acts as a signal and the cognate receptor (CX3CR1).

ICAM-3 is significantly lost from apoptotic leukocyte surfaces when microparticles are shed, which are strongly chemoattractive in a manner that is specific and relies on the ICAM-3. The study establishes ICAM-3 as a crucial adhesion molecule taking part in immune synapse formation and provides new information on the mechanisms of chemoattraction of phagocytes to apoptotic cells and identifies ICAM-3 as a significant adhesion molecule during phagocytosis.

ICAM-3 MA4 monoclonal antibodies inhibit the interaction between phagocytes and apoptotic cells, and they are reactive to the membrane-distal domains of ICAM-3. Novel monoclonal antibodies were raised from mice that had been inoculated with ICAM-3-transferred human embryonic kidney (HEK) cells. The antibodies were screened using the ELISA test, against recombinant ICAM-3 (domains 1 and 2)-Fc fusion protein. CD14-Fc was used as a control. 17 anti-ICAM-3 monoclonal antibodies were identified to be specific for domains 1 and 2 of the ICAM-3. The anti-ICAM-3 monoclonal antibodies were screened to single out MA4 which significantly inhibits phagocytosis by preventing the interaction.

A variety of anti-ICAM-3 monoclonal antibodies, particularly the MA4 that blocks apoptotic cell clearance by macrophages and apoptotic human leukocytes that are deficient or normal for ICAM-3 were used for the study.

Mutu Burkitt's lymphoma (BL) cells were induced to carry out apoptosis by the use of Ultra-Violet rays (100mJ/cm2) and co-cultured with THP-1 phagocytes in the presence of the MA4 antibodies.

The percentage of THP-1 phagocytes involved in the tethering to apoptotic cells and the phagocytosis process was determined using light microscopy after Giemsa/Jenner staining. Four tries were done for each of the three experiments and the data obtained was used to calculate the mean and standard deviation (Peter et al,2008). The data used was normalized to the co-culture free of monoclonal antibodies.

Discussion

The results of the study confirmed that MA4 is a novel anti-ICAM-3 monoclonal antibody with the ability to react with a likely linear neo-epitope in the ICAM-3 domains 1 and 2, which results in the inhibition of the interaction between phagocytes and apoptotic cells, and as a result, prevents phagocytosis. Apoptotic cells with an ICAM-3 deficiency displayed a significant level of exclusion by macrophages and were, therefore, not eliminated. ICAM-3 deficient apoptotic cells exhibited a lowered ability to interact with THP-1 phagocytes. Similar results were obtained with primary human monocyte-derived macrophages. Analysis of programmed cell death indicates that both populations of macrophages and apoptotic cells were depleted with the same kinetics irrespective of the expression of ICAM-3 on the surface of apoptotic cells. In the experiment where phagocytes and apoptotic cells were incubated together in the presence of MA4 monoclonal antibodies, only the cells capable of expressing ICAM-3 had their recognition inhibited, which solidifies the specificity of MA4 and its effects.

The results obtained also showed that apoptotic cell-associated ICAM-3 mediates tethering to phagocytes as a ligand. ICAM-3 levels were also found to drop significantly during the process of apoptosis due to the shedding in microparticles. The decrease in the lymphocyte ICAM-3 levels to cell volume reduction also supports the theory of loss of ICAM-3 levels explaining the loss of ICAM-3. The decline in cell volumes due to apoptosis occurs through, among other mechanisms, the release of blebs to apoptotic microparticles. Dying B cells have also been observed to release microparticles with the accompaniment of losing cell surface molecules which are chemoattractive for macrophages (Savill &Haslett,2002).Microparticles from Mutu BL cells have been found to contain chemoattractive properties that can draw macrophages to cell death sites.

The chemo attraction process is partly reliant on the chemokine CX3CL1 and its receptor CX3CR1 since most times, and recruitment requires an adhesion molecule and a chemokine. Data obtained also revealed a potential for microparticles was breaking off BL cells to promote the movement of phagocytes. Additionally, microparticles released from ICAM-3-deficient BL cells were significantly less potent. Chemotaxis was ruled out, and it was concluded that microparticles from ICAM-3-replete cells promoted chemokinesis instead. Without a gradient, phagocyte movement decreased significantly.

The study suggests that apoptotic cell clearance occurs through the development of a complex phagocytic synapse between the dying cells and the phagocytes that are similar in concept to the ICAM-3-containing immune synapse between T cells and antigen-presenting cells. The role of the IgSF in the apoptotic cell clearance process has been made more explicit, and it may make it possible to identify the specific receptor that recognizes apoptotic cell-associated ICAM-3. The research results illustrate that microparticles have the capability of exerting anti-inflammatory effects on macrophages irrespective of their ICAM-3 expression. The results also suggest the possibility of using ICAM-3 as a target to facilitate the recruitment of monocytes and macrophages to sites of possible leukocyte cell death.

Recent hypotheses state that apoptotic cell-derived

chemoattractant may have the ability to recruit monocytes to tumors where they play a crucial role in driving tumorigenesis as tumor-associated macrophages. The concept may be of importance in cases of non-Hodgkin's lymphoma, where high levels of apoptosis and numerous macrophages coexist. With the study, it may be possible to manipulate the developments to find ways of labeling carcinoma cells according to their proteins for them to have them attract phagocytes. Natural cancer cell elimination on a level enough to beat their proliferation and multiplication will be e better way of beating cancer without killing healthy cells and suppressing the immunity of the patient.

References

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Peter C, Waibel M, Radu CG, Yang LV, Witte ON, Schulze-Osthoff K et al. Migration to apoptotic "find-me" signals is mediated via the phagocyte receptor G2A. J Biol Chem 2008; 283: 5296-5305.

Duvall E, Wyllie AH, Morris RG. Macrophage recognition of cells undergoing programmed cell deat(apoptosis). Immunology 1985; 56: 351-358.

Tabas I. Macrophage death and defective inflammation resolution in atherosclerosis. Nat Rev Immunol 2010; 10: 36-46.