https://www.cell.com/cell/pdf/S0092-8674(05)00869-X.pdf

Cell-Surface Calreticulin Initiates Clearance of Viable or Apoptotic Cells through trans-Activation of LRP on the Phagocyte

It is currently thought that immune responses are initiated by pathogen-associated molecular patterns or by tissue-derived danger/alarm signals. Here, we propose that these two groups of molecules might not be mutually exclusive. Many of them might be part of an evolutionarily ancient alert system in which the hydrophobic portions of biological molecules act, when exposed, as universal damage-associated molecular patterns to initiate repair, remodelling and immunity.

Hydrophobicity: an ancient damage-associated molecular pattern that initiates innate immune responses

During programmed cell death in multicellular organisms, a large number of cells are engulfed by macrophages, thus avoiding the release of noxious materials from the dying cells. These 'apoptotic' cells expose phosphatidylserine (PS) on their surface as an 'eat-me' signal. Miyanishi et al. show that the receptors Tim4 and Tim1 are implicated in phagocyte recognition of PS, while Park et al. show that the BAI1 protein is a receptor for PS in mammalian macrophages. Apoptotic cells expose phosphatidylserine as an 'eat-me' signal for macrophages. This paper shows that the receptors Tim4 and Tim1 are implicated in phagocyte recognition of phosphatidylserine. In programmed cell death, a large number of cells undergo apoptosis, and are engulfed by macrophages to avoid the release of noxious materials from the dying cells1,2. In definitive erythropoiesis, nuclei are expelled from erythroid precursor cells and are engulfed by macrophages. Phosphatidylserine is exposed on the surface of apoptotic cells3 and on the nuclei expelled from erythroid precursor cells4; it works as an ‘eat me’ signal for phagocytes5,6. Phosphatidylserine is also expressed on the surface of exosomes involved in intercellular signalling7. Here we established a library of hamster monoclonal antibodies against mouse peritoneal macrophages, and found an antibody that strongly inhibited the phosphatidylserine-dependent engulfment of apoptotic cells. The antigen recognized by the antibody was identified by expression cloning as a type I transmembrane protein called Tim4 (T-cell immunoglobulin- and mucin-domain-containing molecule; also known as Timd4)8. Tim4 was expressed in Mac1+ cells in various mouse tissues, including spleen, lymph nodes and fetal liver. Tim4 bound apoptotic cells by recognizing phosphatidylserine via its immunoglobulin domain. The expression of Tim4 in fibroblasts enhanced their ability to engulf apoptotic cells. When the anti-Tim4 monoclonal antibody was administered into mice, the engulfment of apoptotic cells by thymic macrophages was significantly blocked, and the mice developed autoantibodies. Among the other Tim family members, Tim1, but neither Tim2 nor Tim3, specifically bound phosphatidylserine. Tim1- or Tim4-expressing Ba/F3 B cells were bound by exosomes via phosphatidylserine, and exosomes stimulated the interaction between Tim1 and Tim4. These results indicate that Tim4 and Tim1 are phosphatidylserine receptors for the engulfment of apoptotic cells, and may also be involved in intercellular signalling in which exosomes are involved.

/pdf/identification-of-tim4-as-a-phosphatidylserine-receptor-3fb9wgkre3.pdf

Identification of Tim4 as a phosphatidylserine receptor.

Tissue-factor–bearing microvesicles arise from lipid rafts and fuse with activated platelets to initiate coagulation

Phosphatidylserine (PS) is the most abundant negatively charged phospholipid in eukaryotic membranes. PS directs the binding of proteins that bear C2 or gamma-carboxyglutamic domains and contributes to the electrostatic association of polycationic ligands with cellular membranes. Rather than being evenly distributed, PS is found preferentially in the inner leaflet of the plasma membrane and in endocytic membranes. The loss of PS asymmetry is an early indicator of apoptosis and serves as a signal to initiate blood clotting. This review discusses the determinants and functional implications of the subcellular distribution and membrane topology of PS.

The distribution and function of phosphatidylserine in cellular membranes.

Although different macrophages exploit different cell surface receptors to recognize apoptotic lymphocytes, indirect evidence suggested that the phosphatidylserine (PS) that appears on the surface of lymphocytes undergoing apoptosis participates in specific recognition by all types of macrophages. To test this possibility directly, annexin V, a protein that specifically binds to PS, was used to mask this phospholipid on the apoptotic cell surface. Preincubation of apoptotic lymphocytes with annexin V blocked phagocytosis by elicited mouse peritoneal macrophages, macrophages of the mouse J774 cell line and mouse bone marrow macrophages. Similarly, annexin V was able to inhibit phagocytosis of lipid-symmetric erythrocytes, another target cell upon which PS is exposed. Together these results demonstrate directly that macrophages of all types depend on the PS exposed on the surface of apoptotic lymphocytes for recognition and phagocytosis.

/pdf/exposure-of-phosphatidylserine-is-a-general-feature-in-the-2fceo9a6po.pdf

Exposure of phosphatidylserine is a general feature in the phagocytosis of apoptotic lymphocytes by macrophages.

Cells generally maintain an asymmetric distribution of phospholipids across the plasma membrane bilayer, restricting the phospholipid, phosphatidylserine (PS), to the inner leaflet of the plasma membrane. When cells undergo apoptosis, this asymmetric transbilayer distribution is lost, bringing PS to the surface where it acts as a signal for engulfment by phagocytes. The fluorescent dye merocyanine 540 specifically stains the plasma membrane of apoptotic cells which have lost their asymmetric distribution of phospholipids. However, it also stains non-apoptotic macrophages, suggesting that phospholipid asymmetry may not be maintained in these cells, and thus that they may express PS on their surface. Here, the PS-binding protein, annexin V, was used to show that in fact normal macrophages do express PS on their surface. Furthermore, pre-treating macrophages with annexin V was found to inhibit phagocytosis of apoptotic thymocytes and thymocytes on which PS expression was artificially induced, but did not inhibit phagocytosis of latex beads or Fc receptor-mediated phagocytosis of opsonized erythrocytes. These results indicate that PS is constitutively expressed on the surface of macrophages and is functionally significant for the phagocytosis of PS-expressing target cells.

/pdf/surface-expression-of-phosphatidylserine-on-macrophages-is-1gmmsg4z3b.pdf

Surface expression of phosphatidylserine on macrophages is required for phagocytosis of apoptotic thymocytes.

HIV-1 is an enveloped retrovirus that acquires its outer membrane as the virion exits the cell. Because of the association of apoptosis with the progression of AIDS, HIV-1-infected T cells or macrophages might be expected to express elevated levels of surface phosphatidylserine (PS), a hallmark of programmed cell death. Virions produced by these cells would also be predicted to have PS on the surface of their envelopes. In this study, data are presented that support this hypothesis and suggest that PS is required for macrophage infection. The PS-specific protein annexin V was used to enrich for virus particles and to inhibit HIV-1 replication in primary macrophages, but not T cells. HIV-1 replication was also significantly inhibited with vesicles consisting of PS, but not phosphatidylcholine. PS is specifically required for HIV-1 infection because viruses pseudotyped with vesicular stomatitis virus G and amphotropic murine leukemia virus envelopes were not inhibited by PS vesicles or annexin V. These data indicate that PS is an important cofactor for HIV-1 infection of macrophages.

Phosphatidylserine on HIV envelope is a cofactor for infection of monocytic cells.

Expression of the aminophospholipid phosphatidylserine (PS) on the surface of apoptotic lymphocytes and lipid-symmetric erythrocytes triggers their phagocytosis by macrophages. Phagocytosis by both activated and unactivated macrophages, which utilize different recognition systems, can be blocked by certain monoclonal antibodies directed against the LPS receptor, CD14. Here we investigate the requirement for CD14 in the phagocytosis of both apoptotic thymocytes and lipid-symmetric erythrocytes by both activated and unactivated macrophages. We show that phagocytosis of lipid-symmetric erythrocytes by both activated and unactivated macrophages is completely abolished when CD14 is removed from macrophages by cleaving its glycosylphosphatidylinositol tether with phospholipase C. This treatment also substantially reduces phagocytosis of apoptotic lymphocytes by both types of macrophages. Unactivated LR-9 mouse macrophages which are deficient in CD14 expression are completely unable to phagocytose either apoptotic thymocytes or lipid-symmetric erythrocytes. These results argue that CD14 is an absolute requirement for the phagocytosis of lipid-symmetric erythrocytes by both activated and unactivated macrophages, despite their different recognition systems, that CD14 contributes at least substantially to the phagocytosis of apoptotic lymphocytes by both activated and unactivated macrophages, and that activated macrophages may also possess an alternate, CD14-independent mechanism for phagocytosis of apoptotic lymphocytes.

/pdf/cd14-is-a-component-of-multiple-recognition-systems-used-by-4agfw7oy3k.pdf

CD14 is a component of multiple recognition systems used by macrophages to phagocytose apoptotic lymphocytes.

Expression of phosphatidylserine (PS) on the surface of both macrophages and their apo- ptotic targets is required for efficient phagocytosis. Monocytes, the precursors of macrophages, do not express PS on their surface and do not efficiently phagocytose apoptotic cells. We report here that PS appears on the surface of both human mono- cytic U937 cells and primary human monocytes as they differentiate in culture and acquire the ability to phagocytose apoptotic thymocytes. Phagocyto- sis was blocked by pretreating either the apoptotic target or the phagocyte with annexin V to mask PS and was CD14-dependent. Expression of PS, like other events characteristic of differentiating mono- cytes such as Mac-1 expression, was independent of the agent used to induce differentiation and was insensitive to the addition of caspase inhibitors. These results demonstrate that PS is expressed on monocytes as part of their differentiation program and is independent of apoptosis. J. Leukoc. Biol. 74: 000-000; 2003.

https://jlb.onlinelibrary.wiley.com/doi/pdf/10.1189/jlb.0902433

Melissa K. Callahan

Papers

Exposure of phosphatidylserine is a general feature in the phagocytosis of apoptotic lymphocytes by macrophages.

Surface expression of phosphatidylserine on macrophages is required for phagocytosis of apoptotic thymocytes.

Phosphatidylserine on HIV envelope is a cofactor for infection of monocytic cells.

CD14 is a component of multiple recognition systems used by macrophages to phagocytose apoptotic lymphocytes.

Phosphatidylserine expression and phagocytosis of apoptotic thymocytes during differentiation of monocytic cells