Donna L. Bratton

Macrophages that have ingested apoptotic cells in vitro inhibit proinflammatory cytokine production through autocrine/paracrine mechanisms involving TGF-beta, PGE2, and PAF.

Apoptosis in vivo is followed almost inevitably by rapid uptake into adjacent phagocytic cells, a critical process in tissue remodeling, regulation of the immune response, or resolution of inflammation. Phagocytosis of apoptotic cells by macrophages has been suggested to be a quiet process that does not lead to production of inflammatory mediators. Here we show that phagocytosis of apoptotic neutrophils (in contrast to immunoglobulin G-opsonized apoptotic cells) actively inhibited the production of interleukin (IL)-1beta, IL-8, IL-10, granulocyte macrophage colony-stimulating factor, and tumor necrosis factor-alpha, as well as leukotriene C4 and thromboxane B2, by human monocyte-derived macrophages. In contrast, production of transforming growth factor (TGF)-beta1, prostaglandin E2, and platelet-activating factor (PAF) was increased. The latter appeared to be involved in the inhibition of proinflammatory cytokine production because addition of exogenous TGF-beta1, prostaglandin E2, or PAF resulted in inhibition of lipopolysaccharide-stimulated cytokine production. Furthermore, anti-TGF-beta antibody, indomethacin, or PAF receptor antagonists restored cytokine production in lipopolysaccharide-stimulated macrophages that had phagocytosed apoptotic cells. These results suggest that binding and/or phagocytosis of apoptotic cells induces active antiinflammatory or suppressive properties in human macrophages. Therefore, it is likely that resolution of inflammation depends not only on the removal of apoptotic cells but on active suppression of inflammatory mediator production. Disorders in either could result in chronic inflammatory diseases.

A receptor for phosphatidylserine-specific clearance of apoptotic cells

The culmination of apoptosis in vivo is phagocytosis of cellular corpses. During apoptosis, the asymmetry of plasma membrane phospholipids is lost, which exposes phosphatidylserine externally. The phagocytosis of apoptotic cells can be inhibited stereospecifically by phosphatidylserine and its structural analogues, but not by other anionic phospholipids, suggesting that phosphatidylserine is specifically recognized. Using phage display, we have cloned a gene that appears to recognize phosphatidylserine on apoptotic cells. Here we show that this gene, when transfected into B and T lymphocytes, enables them to recognize and engulf apoptotic cells in a phosphatidylserine-specific manner. Flow cytometric analysis using a monoclonal antibody suggested that the protein is expressed on the surface of macrophages, fibroblasts and epithelial cells; this antibody, like phosphatidylserine liposomes, inhibited the phagocytosis of apoptotic cells and, in macrophages, induced an anti-inflammatory state. This candidate phosphatidylserine receptor is highly homologous to genes of unknown function in Caenorhabditis elegans and Drosophila melanogaster, suggesting that phosphatidylserine recognition on apoptotic cells during their removal by phagocytes is highly conserved throughout phylogeny.

The role of phosphatidylserine in recognition of apoptotic cells by phagocytes

Exposure of phosphatidylserine on the outer leaflet of the plasma membrane is a surface change common to many apoptotic cells. Normally restricted to the inner leaflet, phosphatidylserine appears as a result of decreased aminophospholipid translocase activity and activation of a calcium-dependent scramblase. Phosphatidylserine exposure has several potential biological consequences, one of which is recognition and removal of the apoptotic cell by phagocytes. It is still not clear which receptors mediate PS recognition on apoptotic cells; however, several interesting candidates have been proposed. These include the Class B scavenger and thrombospondin receptor CD36, an oxLDL receptor (CD68), CD14, annexins, β2 glycoprotein I, gas-6 and a novel activity expressed on macrophages stimulated with digestible particles such as β-glucan. Whether PS is the sole ligand recognized by phagocytes or whether it associated with other molecules to form a complex ligand remains to be determined.

Phosphatidylserine (PS) induces PS receptor–mediated macropinocytosis and promotes clearance of apoptotic cells

Efficient phagocytosis of apoptotic cells is important for normal tissue development, homeostasis, and the resolution of inflammation. Although many receptors have been implicated in the clearance of apoptotic cells, the roles of these receptors in the engulfment process have not been well defined. We developed a novel system to distinguish between receptors involved in tethering of apoptotic cells versus those inducing their uptake. Our results suggest that regardless of the receptors engaged on the phagocyte, ingestion does not occur in the absence of phosphatidylserine (PS). Further, recognition of PS was found to be dependent on the presence of the PS receptor (PSR). Both PS and anti-PSR antibodies stimulated membrane ruffling, vesicle formation, and “bystander” uptake of cells bound to the surface of the phagocyte. We propose that the phagocytosis of apoptotic cells requires two events: tethering followed by PS-stimulated, PSR-mediated macropinocytosis.

Apoptotic cell removal

Ingestion by professional or amateur phagocytes is the fate of most cells that undergo apoptosis. Studies in both Caenorhabditis elegans and mammals are now converging to reveal some of the key mechanisms and consequences of this removal process. At least seven corpse removal genes in nematodes have mammalian equivalents, and represent elements of signaling pathways involved in uptake. In mammals, a wide variety of apoptotic cell recognition receptors has been implicated and appears to be divided into two categories, involved in tethering the apoptotic cell or triggering an uptake mechanism related to macropinocytosis. Apoptotic cell removal is normally efficient and non-inflammatory. By contrast, the process may become subverted by parasites to yield a more favorable growth environment, or in other cases lead to fibrosis. Removal may also clinch the apoptotic process itself in cells not yet completely committed to death.

Phagocyte receptors for apoptotic cells: recognition, uptake, and consequences

Apoptosis, a genetically programmed cell death conserved throughout phylogeny, provides a counterbalance to mitosis in the regulation of tissue growth and homeostasis (1, 2). Interest in and knowledge of the mechanisms mediating the induction and execution of the death program have blossomed in the last decade and a half. Engulfment of apoptotic bodies and debris represents the denouement of the death program for most cells in multicellular organisms. Macrophages and other cells manifesting their primitive phagocytic potential clear apoptotic bodies from tissues, preventing their lysis and the consequent release of toxic or immunogenic intracellular components. By inducing the release of mediators such as TGF-β, IL-10, prostaglandin E2 (PGE2), and others, clearance of apoptotic cells also sets up an anti-inflammatory milieu within the tissue (reviewed in refs. 3, 4). However, phagocytic clearance is not merely a silent process with respect to inflammation and immunity but is also actively anti-inflammatory. In spite of its critical importance to tissue and host homeostasis, clearance of apoptotic cells is poorly understood, and studies devoted to elucidating the mechanisms mediating recognition and engulfment have lagged behind those probing the induction and execution of the death process itself. This situation is finally changing as we begin to unravel the mechanisms mediating clearance of apoptotic cells. Many of the macrophage receptors that contribute to apoptotic cell recognition are critical players in innate immunity. These include certain lectins and integrins, the class A and class B scavenger receptors (see Platt and Gordon, this Perspective series, ref. 5; and Krieger, this series, ref. 6), receptors for oxidized LDL, including CD68 and lectin-like oxidized LDL receptor-1 (LOX-1), some of the receptors for complement-derived proteins, and the endotoxin receptor CD14 (reviewed in ref. 7). Paradoxically, however, when microbial organisms or their products are engulfed via these receptors, inflammation results, and in many cases, acquired immunity is stimulated. In contrast, uptake of apoptotic cells is generally noninflammatory and thereby avoids activation of an acquired immune response. Our task is to understand and explain these diametrically opposed reactions to stimulation of the same receptors.

Appearance of Phosphatidylserine on Apoptotic Cells Requires Calcium-mediated Nonspecific Flip-Flop and Is Enhanced by Loss of the Aminophospholipid Translocase*

Phosphatidylserine (PS), ordinarily sequestered in the plasma membrane inner leaflet, appears in the outer leaflet during apoptosis, where it triggers non-inflammatory phagocytic recognition of the apoptotic cell. The mechanism of PS appearance during apoptosis is not well understood but has been associated with loss of aminophospholipid translocase activity and nonspecific flip-flop of phospholipids of various classes. The human leukemic cell line HL-60, the T cell line Jurkat, and peripheral blood neutrophils, undergoing apoptosis induced either with UV irradiation or anti-Fas antibody, were probed in the cytofluorograph for (i) surface PS using fluorescein isothiocyanate-labeled annexin V, (ii) PS uptake by the aminophospholipid translocase using [6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino] caproyl] (NBD)-labeled PS, (iii) nonspecific uptake of phospholipids (as a measure of transbilayer flip-flop) using NBD-labeled phosphatidylcholine, and (iv) the appearance of hypodiploid DNA. In all three types of cells undergoing apoptosis, the appearance of PS followed loss of aminophospholipid translocase and was accompanied by nonspecific phospholipid flip-flop. Importantly, however, in the absence of extracellular calcium, the appearance of PS was completely inhibited despite DNA fragmentation and loss of aminophospholipid translocase activity, the latter demonstrating that loss of the translocase is insufficient for PS appearance during apoptosis. Furthermore, while both the appearance of PS and nonspecific phospholipid uptake demonstrated identical extracellular calcium requirements with an ED50 of nearly 100 μm, the magnitude of PS appearance depended on the level of aminophospholipid translocase activity. Taken together, the data strongly suggest that while nonspecific flip-flop is the driving event for PS appearance in the plasma membrane outer leaflet, aminophospholipid translocase activity ultimately modulates its appearance.

Elastase-mediated phosphatidylserine receptor cleavage impairs apoptotic cell clearance in cystic fibrosis and bronchiectasis

Cystic fibrosis is characterized by an early and sustained influx of inflammatory cells into the airways and by release of proteases. Resolution of inflammation is normally associated with the orderly removal of dying apoptotic inflammatory cells through cell recognition receptors, such as the phosphatidylserine receptor, CD36, and alpha v integrins. Accordingly, removal of apoptotic inflammatory cells may be impaired in persistent inflammatory responses such as that seen in cystic fibrosis airways. Examination of sputa from cystic fibrosis and non-cystic fibrosis bronchiectasis patients demonstrated an abundance of apoptotic cells, in excess of that seen in patients with chronic bronchitis. In vitro, cystic fibrosis and bronchiectasis airway fluid directly inhibited apoptotic cell removal by alveolar macrophages in a neutrophil elastase-dependent manner, suggesting that elastase may impair apoptotic cell clearance in vivo. Flow cytometry demonstrated that neutrophil elastase cleaved the phosphatidylserine receptor, but not CD36 or CD32 (Fc gamma RII). Cleavage of the phosphatidylserine receptor by neutrophil elastase specifically disrupted phagocytosis of apoptotic cells, implying a potential mechanism for delayed apoptotic cell clearance in vivo. Therefore, defective airway clearance of apoptotic cells in cystic fibrosis and bronchiectasis may be due to elastase-mediated cleavage of phosphatidylserine receptor on phagocytes and may contribute to ongoing airway inflammation.

Differential Effects of Apoptotic Versus Lysed Cells on Macrophage Production of Cytokines: Role of Proteases

Granulocytes undergoing apoptosis are recognized and removed by phagocytes before their lysis. The release of their formidable arsenal of proteases and other toxic intracellular contents into tissues can create significant damage, prolonging the inflammatory response. Binding and/or uptake of apoptotic cells by macrophages inhibits release of proinflammatory cytokines by mechanisms that involve anti-inflammatory mediators, including TGF-β. To model the direct effects of necrotic cells on macrophage cytokine production, we added lysed or apoptotic neutrophils and lymphocytes to mouse and human macrophages in the absence of serum to avoid complement activation. The results confirmed the ability of lysed neutrophils, but not lymphocytes, to significantly stimulate production of macrophage-inflammatory protein 2 or IL-8, TNF-α, and IL-10. Concomitantly, induction of TGF-β1 by lysed neutrophils was significantly lower than that observed for apoptotic cells. The addition of selected serine protease inhibitors and anti-human elastase Ab markedly reduced the proinflammatory effects, the lysed neutrophils then behaving as an anti-inflammatory stimulus similar to intact apoptotic cells. Separation of lysed neutrophils into membrane and soluble fractions showed that the neutrophil membranes behaved like apoptotic cells. Thus, the cytokine response seen when macrophages were exposed to lysed neutrophils was largely due to liberated proteases. Therefore, we suggest that anti-inflammatory signals can be given by PtdSer-containing cell membranes, whether from early apoptotic, late apoptotic, or lysed cells, but can be overcome by proteases liberated during lysis. Therefore, the outcome of an inflammatory reaction and the potential immunogenicity of Ags within the damaged cell will be determined by which signals predominate.

The phosphatidylserine receptor: a crucial molecular switch?

The uptake and removal of necrotic or lysed cells involves inflammation and an immune response, due in part to processes that involve members of the collectin family, surface calreticulin and CD91. Clearance of apoptotic cells, by contrast, does not induce either inflammation or immunity. Could the phosphatidylserine receptor be the molecular switch that determines what the outcome will be?