Programmed cell clearance : mechanisms and consequences of phagocytosis of apoptotic cells

Programmed cell clearance involves the engulfment of apoptotic cells by professional phagocytes (macrophages) or neighboring cells. During normal development and tissue regeneration a massive number of cells undergo apoptosis. The subsequent removal of the cell corpses by phagocytes is crucial to prevent disintegration of the cell membrane and leakage of noxious constituents into surrounding tissues. Efficient phagocytosis requires recognition or eat-me signals, soluble bridging molecules and phagocytosis receptors. Phosphatidylserine (PS) is externalized on the plasma membrane during apoptosis and is one of the most well-studied recognition signals for phagocytosis.

The importance of PS exposure was addressed in the present thesis using different in vitro models. We found that plasma membrane blebbing could be dissociated from other features of the apoptotic program. PS-positive cells that failed to display membrane blebbing during apoptosis were observed to escape engulfment by macrophages. However, the PS-binding bridging molecule milk fat globule epidermal growth factor 8 (MFG-E8) increased the efficiency of phagocytosis of non-blebbing apoptotic cells. To further study the importance of PS in the clearance of neutrophils we established a model of macrophage-induced PS exposure. Macrophage-differentiated PLB-985 cells triggered caspase- and NADPH oxidase-independent PS externalization in primary human neutrophils. These neutrophils exhibited similar levels of PS exposure as neutrophils undergoing constitutive apoptosis. However, the phagocytosis of PLB-985-co-cultured neutrophils by human monocyte-derived macrophages (HMDM) was considerably lower, indicating that PS externalization alone is not sufficient for macrophage disposal of neutrophils. The addition of recombinant MFG-E8 restored macrophage engulfment of these cells. Moreover, PLB-985-co-cultured neutrophils displayed significantly lower surface expression and release of annexin I compared to spontaneous apoptotic neutrophils.

Phagocytosis of macrophage-co-cultured neutrophils was promoted when annexin I-enriched cell culture medium was added, and this process was blocked by Boc1 (formyl peptide receptor/lipoxin receptor antagonist). A role for annexin I was also found in the engulfment of pre-apoptotic Jurkat cells briefly treated with agonistic anti-Fas antibody or recombinant Fas ligand. These cells secreted annexin I, and were ingested prior to the occurrence of common biomarkers of apoptosis, including PS exposure. Moreover, Boc1 markedly attenuated their engulfment. Similar findings were obtained when using primary human T cells. Furthermore, pre-apoptotic Jurkat cells induced lower macrophage production of TNF-α and higher production of IL-10 in comparison to apoptotic target cells. Finally, the interaction of HMDM and mesoporous silica particles was examined. Efficient and active internalization of mesoporous silica particles of different sizes was observed and appeared to occur through a process of endocytosis. Uptake of mesoporous silica particles did not affect viability of human macrophages, or the function of these cells, including the ingestion of various classes of apoptotic or opsonized target cells. In summary, these studies contribute to our understanding of the important physiological process of programmed cell clearance and may have implications for chronic inflammation and autoimmune disease. Studies of the interaction of nanomaterials with phagocytes are also relevant for the development of these materials for biomedical applications.

List of scientific papers

I. Witasp E, Uthaisang W, Elenström-Magnusson C, Hanayama R, Tanaka M, Nagata S, Orrenius S, Fadeel B (2007). "Bridge over troubled water: milk fat globule epidermal growth factor 8 promotes human monocyte-derived macrophage clearance of non-blebbing phosphatidylserine-positive target cells." Cell Death Differ 14(5): 1063-5. Epub 2007 Jan 26
https://pubmed.ncbi.nlm.nih.gov/17256011

II. Jitkaew S, Witasp E, Zhang S, Kagan VE, Fadeel B (2008). "Induction of caspase- and reactive oxygen species-independent phosphatidylserine externalization in primary human neutrophils: role in macrophage recognition and engulfment." J Leukoc Biol Dec 23: Epub ahead of print
https://pubmed.ncbi.nlm.nih.gov/19106181

III. Zhang S, Witasp E, Lauwen M, Fadeel B (2008). "Brief cross-linking of Fas/APO-1 (CD95) triggers engulfment of pre-apoptotic target cells." FEBS Lett 582(23-24): 3501-8. Epub 2008 Sep 18
https://pubmed.ncbi.nlm.nih.gov/18804468

IV. Witasp E., Kagan V., Fadeel B (2008). "APPENDIX I: Programmed cell clearance: molecular mechanisms and role in autoimmune disease, chronic inflammation, and anti-cancer immune responses." Curr. Immunol. Rev 4: 53-69