Removal of apoptotic cells is essential for maintenance of tissue homeostasis, organogenesis, remodeling, development, and maintenance of the immune system, protection against neoplasia, and resolution of inflammation. The mechanisms of this removal involve recognition of the apoptotic cell surface and initiation of phagocytic uptake into a variety of cell types. Here we provide evidence that C1q and mannose binding lectin (MBL), a member of the collectin family of proteins, bind to apoptotic cells and stimulate ingestion of these by ligation on the phagocyte surface of the multifunctional protein, calreticulin (also known as the cC1qR), which in turn is bound to the endocytic receptor protein CD91, also known as the α-2-macroglobulin receptor. Use of these proteins provides another example of apoptotic cell clearance mediated by pattern recognition molecules of the innate immune system. Ingestion of the apoptotic cells through calreticulin/CD91 stimulation is further shown to involve the process of macropinocytosis, implicated as a primitive and relatively nonselective uptake mechanism for C1q- and MBL-enhanced engulfment of whole, intact apoptotic cells, as well as cell debris and foreign organisms to which these molecules may bind.

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.

Removal of cells dying by apoptosis is essential to normal development, maintenance of tissue homeostasis, and resolution of inflammation. Surfactant protein A (SP-A) and surfactant protein D (SP-D) are high abundance pulmonary collectins recently implicated in apoptotic cell clearance in vitro. Other collectins, such as mannose-binding lectin and the collectin-like C1q, have been shown to bind to apoptotic cells and drive ingestion through interaction with calreticulin and CD91 on the phagocyte in vitro. However, only C1q has been shown to enhance apoptotic cell uptake in vivo. We sought to determine the relative importance of SP-A, SP-D, and C1q in pulmonary clearance of apoptotic cells using knockout and overexpressing mice, and to determine the role of calreticulin and CD91 in this process. SP-A, SP-D, and C1q all enhanced apoptotic cell ingestion by resident murine and human alveolar macrophages in vitro. However, only SP-D altered apoptotic cell clearance from the naive murine lung, suggesting that SP-D plays a particularly important role in vivo. Similar to C1q and mannose-binding lectin, SP-A and SP-D bound to apoptotic cells in a localized, patchy pattern and drove apoptotic cell ingestion by phagocytes through a mechanism dependent on calreticulin and CD91. These results suggest that the entire collectin family of innate immune proteins (including C1q) works through a common receptor complex to enhance removal of apoptotic cells, and that collectins are integral, organ-specific components of the clearance machinery.

Cells undergoing apoptosis are efficiently located and engulfed by phagocytes. The mechanisms by which macrophages, the professional scavenging phagocytes of apoptotic cells, are attracted to sites of apoptosis are poorly defined. Here we show that CX3CL1/fractalkine, a chemokine and intercellular adhesion molecule, is released rapidly from apoptotic lymphocytes, via caspase-and Bcl-2-regulated mechanisms, to attract macrophages. Effective chemotaxis of macrophages to apoptotic lymphocytes is dependent on macrophage fractalkine receptor, CX3CR1. CX3CR1 deficiency caused diminished recruitment of macrophages to germinal centers of lymphoid follicles, sites of high-rate B-cell apoptosis. These results provide the first demonstration of chemokine/chemokine-receptor activity in the navigation of macrophages toward apoptotic cells and identify a mechanism by which macrophage infiltration of tissues containing apoptotic lymphocytes is achieved. (Blood. 2008;112:5026-5036) IntroductionWhen apoptosis occurs at high rates in mammalian tissues, apoptotic cells are almost invariably encountered in situ in association with macrophages. 1 These professional scavengers are attracted to the dying cells and engage in their safe, nonphlogistic disposal by phagocytosis. Examples of this innate immune response to dying cells are readily apparent during normal organogenesis, in normal adult tissues, such as the germinal centers of lymphoid follicles, in inflammatory responses, and in pathologic conditions including tumors. The efficient clearance of apoptotic cells by phagocytes is a homeostatic mechanism that militates against histotoxic, proinflammatory, or immunogenic effects that may result from persistence of apoptotic cells. [1][2][3] In recent years, much progress has been made in improving our understanding of the molecular mechanisms underlying the interactions between apoptotic cells and macrophages and the immunologic implications of those interactions. [1][2][3][4][5][6] Before the tethering/ engulfment phases of macrophage-mediated apoptotic-cell clearance, phagocytes are required to navigate effectively to sites of apoptosis. Active release of chemoattractant ("find-me") signals from apoptotic cells at an early stage after engagement of the cell-death program would be predicted to underpin this process, but knowledge of the molecules involved is currently limited. Lysophosphatidylcholine (LPC) is released from apoptotic cells and functions in soluble form as a chemoattractant for mononuclear phagocytes. 7 Significantly, no chemokine family members have previously been implicated in this chemotactic process. Here we show that the chemokine and adhesion molecule CX3CL1, 8,9 also known as neurotactin or fractalkine (FKN), together with its cognate receptor CX3CR1, 10,11 plays an active role in the chemotaxis of macrophages to apoptotic cells. FKN is a type I transmembrane protein, the extracellular portion of which comprises the chemokine domain attached to a mucin stalk. Well known for its roles in inflammatory processe...