Ly6C(+) monocyte efferocytosis and cross-presentation of cell-associated antigens.

Recently it was shown that circulating Ly6C(+) monocytes traffic from tissue to the draining lymph nodes (LNs) with minimal alteration in their overall phenotype. Furthermore, in the steady state, Ly6C(+) monocytes are as abundant as classical dendritic cells (DCs) within the draining LNs, and even more abundant during inflammation. However, little is known about the functional roles of constitutively trafficking Ly6C(+) monocytes. In this study we investigated whether Ly6C(+) monocytes can efferocytose (acquire dying cells) and cross-present cell-associated antigen, a functional property particularly attributed to Batf3(+) DCs. We demonstrated that Ly6C(+) monocytes intrinsically efferocytose and cross-present cell-associated antigen to CD8(+) T cells. In addition, efferocytosis was enhanced upon direct activation of the Ly6C(+) monocytes through its corresponding TLRs, TLR4 and TLR7. However, only ligation of TLR7, and not TLR4, enhanced cross-presentation by Ly6C(+) monocytes. Overall, this study outlines two functional roles, among others, that Ly6C(+) monocytes have during an adaptive immune response.

Mammary epithelial cell phagocytosis downstream of TGF-ß3 is characterized by adherens junction reorganization.

After weaning, during mammary gland involution, milk-producing mammary epithelial cells undergo apoptosis. Effective clearance of these dying cells is essential, as persistent apoptotic cells have a negative impact on gland homeostasis, future lactation and cancer susceptibility. In mice, apoptotic cells are cleared by the neighboring epithelium, yet little is known about how mammary epithelial cells become phagocytic or whether this function is conserved between species. Here we use a rat model of weaning-induced involution and involuting breast tissue from women, to demonstrate apoptotic cells within luminal epithelial cells and epithelial expression of the scavenger mannose receptor, suggesting conservation of phagocytosis by epithelial cells. In the rat, epithelial transforming growth factor-ß (TGF-ß) signaling is increased during involution, a pathway known to promote phagocytic capability. To test whether TGF-ß enhances the phagocytic ability of mammary epithelial cells, non-transformed murine mammary epithelial EpH4 cells were cultured to achieve tight junction impermeability, such as occurs during lactation. TGF-ß3 treatment promoted loss of tight junction impermeability, reorganization and cleavage of the adherens junction protein E-cadherin (E-cad), and phagocytosis. Phagocytosis correlated with junction disruption, suggesting junction reorganization is necessary for phagocytosis by epithelial cells. Supporting this hypothesis, epithelial cell E-cad reorganization and cleavage were observed in rat and human involuting mammary glands. Further, in the rat, E-cad cleavage correlated with increased γ-secretase activity and ß-catenin nuclear localization. In vitro, pharmacologic inhibitors of γ-secretase or ß-catenin reduced the effect of TGF-ß3 on phagocytosis to near baseline levels. However, ß-catenin signaling through LiCl treatment did not enhance phagocytic capacity, suggesting a model in which both reorganization of cell junctions and ß-catenin signaling contribute to phagocytosis downstream of TGF-ß3. Our data provide insight into how mammary epithelial cells contribute to apoptotic cell clearance, and in light of the negative consequences of impaired apoptotic cell clearance during involution, may shed light on involution-associated breast pathologies.

Apoptosis in the lung: induction, clearance and detection.

Apoptosis and other forms of programmed cell death are important contributors to lung pathophysiology. In this brief review, we discuss some of the implications of finding apoptotic cells in the lung and methods for their detection. The balance between induction of apoptosis and the normally highly efficient clearance of such cells shows that these are highly dynamic processes and suggests that abnormalities of apoptotic cell clearance may be an alternative explanation for their detection. Because recognition of apoptotic cells by other lung cells has additional effects on inflammation, immunity, and tissue repair, local responses to the dying cells may also have important consequences in addition to the cell death itself.

Clearance of apoptotic cells by phagocytes.

Phagocytic clearance of apoptotic cells may be considered to consist of four distinct steps: accumulation of phagocytes at the site where apoptotic cells are located; recognition of dying cells through a number of bridge molecules and receptors; engulfment by a unique uptake process; and processing of engulfed cells within phagocytes. Here, we will discuss these individual steps that collectively are essential for the effective removal of apoptotic cells. This will illustrate our relative lack of knowledge about the initial attraction signals, the specific mechanisms of engulfment and processing in comparison to the extensive literature on recognition mechanisms. There is now mounting evidence that clearance defects are responsible for chronic inflammatory disease and contribute to autoimmunity. Therefore, a better understanding of all aspects of the clearance process is required before it can truly be manipulated for therapeutic gain.

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.

C1q and mannose binding lectin engagement of cell surface calreticulin and CD91 initiates macropinocytosis and uptake of apoptotic cells.

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 alpha-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.

Neutrophil maturation and activation determine anatomic site of clearance from circulation.

The long-term disposition of circulating neutrophils and the site of disappearance from circulation remain unclear. We investigated neutrophil localization in mice using (111)In-labeled murine peripheral blood neutrophils, mature bone marrow neutrophils, and peritoneal exudate neutrophils to track in vivo localization of these different cell populations. Infused peripheral neutrophils were found to localize equally between liver and marrow sites by 4 h (31.2 +/- 1.9 vs. 31.9 +/- 1.8%), whereas exudate neutrophils predominantly localized to liver (42.0 +/- 1.1%) and marrow-derived neutrophils to the marrow (65.9 +/- 6.6%) where they were found to localize predominantly in the hematopoietic cords. Stimulation of marrow neutrophils before infusion caused a shift in localization from marrow to liver, and subsequent induction of an inflammatory site after infusion and marrow sequestration led to remobilization of infused marrow neutrophils but not of peripheral neutrophils. These results indicate that the marrow participates in removing neutrophils from circulation, with evidence supporting both storage and perhaps disposal functions. Furthermore, models for circulating neutrophil homeostasis should consider that the site of retention is governed by the maturation and activation states of the cell.

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?

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-beta. 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-alpha, and IL-10. Concomitantly, induction of TGF-beta1 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.

Tumor necrosis factor-alpha activation of the c-Jun N-terminal kinase pathway in human neutrophils. Integrin involvement in a pathway leading from cytoplasmic tyrosine kinases apoptosis.

The intensity and duration of an inflammatory response depends on the balance of factors that favor perpetuation versus resolution. At sites of inflammation, neutrophils adherent to other cells or matrix components are exposed to tumor necrosis factor-alpha (TNFalpha). Although TNFalpha has been implicated in induction of pro-inflammatory responses, it may also inhibit the intensity of neutrophilic inflammation by promoting apoptosis. Since TNFalpha is not only an important activator of the stress-induced pathways leading to p38 MAPk and c-Jun N-terminal kinase (JNK) but also a potent effector of apoptosis, we investigated the effects of TNFalpha on the JNK pathway in adherent human neutrophils and the potential involvement of this pathway in neutrophil apoptosis. Stimulation with TNFalpha was found to result in beta2 integrin-mediated activation of the cytoplasmic tyrosine kinases Pyk2 and Syk, and activation of a three-part MAPk module composed of MEKK1, MKK7, and/or MKK4 and JNK1. JNK activation was attenuated by blocking antibodies to beta2 integrins, the tyrosine kinase inhibitors, genistein, and tyrphostin A9, a Pyk2-specific inhibitor, and piceatannol, a Syk-specific inhibitor. Exposure of adherent neutrophils to TNFalpha led to the rapid onset of apoptosis that was demonstrated by augmented annexin V binding and caspase-3 cleavage. TNFalpha-induced increases in annexin V binding to neutrophils were attenuated by blocking antibodies to beta2 integrins, and the caspase-3 cleavage was attenuated by tyrphostin A9. Hence, exposure of adherent neutrophils to TNFalpha leads to utilization of the JNK-signaling pathways that may contribute to diverse functional responses including induction of apoptosis and subsequent resolution of the inflammatory response.

Loss of phospholipid asymmetry and surface exposure of phosphatidylserine is required for phagocytosis of apoptotic cells by macrophages and fibroblasts.

Removal of apoptotic cells during tissue remodeling or resolution of inflammation is critical to the restoration of normal tissue structure and function. During apoptosis, early surface changes occur, which trigger recognition and removal by macrophages and other phagocytes. Loss of phospholipid asymmetry results in exposure of phosphatidylserine (PS), one of the surface markers recognized by macrophages. However, a number of receptors have been reported to mediate macrophage recognition of apoptotic cells, not all of which bind to phosphatidylserine. We therefore examined the role of membrane phospholipid symmetrization and PS externalization in uptake of apoptotic cells by mouse macrophages and human HT-1080 fibrosarcoma cells by exposing them to cells that had undergone apoptosis without loss of phospholipid asymmetry. Neither mouse macrophages nor HT-1080 cells recognized or engulfed apoptotic targets that failed to express PS, in comparison to PS-expressing apoptotic cells. If, however, their outer leaflets were repleted with the l-, but not the d-, stereoisomer of sn-1,2-PS by liposome transfer, engulfment by both phagocytes was restored. These observations directly demonstrate that loss of phospholipid asymmetry and PS expression is required for phagocyte engulfment of apoptotic cells and imply a critical, if not obligatory, role for PS recognition in the uptake process.

Differential roles for alpha(M)beta(2) integrin clustering or activation in the control of apoptosis via regulation of akt and ERK survival mechanisms.

The role of integrins in leukocyte apoptosis is unclear, some studies suggest enhancement, others inhibition. We have found that beta(2)-integrin engagement on neutrophils can either inhibit or enhance apoptosis depending on the activation state of the integrin and the presence of proapoptotic stimuli. Both clustering and activation of alpha(M)beta(2) delays spontaneous, or unstimulated, apoptosis, maintains mitochondrial membrane potential, and prevents cytochrome c release. In contrast, in the presence of proapoptotic stimuli, such as Fas ligation, TNFalpha, or UV irradiation, ligation of active alpha(M)beta(2) resulted in enhanced mitochondrial changes and apoptosis. Clustering of inactive integrins did not show this proapoptotic effect and continued to inhibit apoptosis. This discrepancy was attributed to differential signaling in response to integrin clustering versus activation. Clustered, inactive alpha(M)beta(2) was capable of stimulating the kinases ERK and Akt. Activated alpha(M)beta(2) stimulated Akt, but not ERK. When proapoptotic stimuli were combined with either alpha(M)beta(2) clustering or activation, Akt activity was blocked, allowing integrin activation to enhance apoptosis. Clustered, inactive alpha(M)beta(2) continued to inhibit stimulated apoptosis due to maintained ERK activity. Therefore, beta(2)-integrin engagement can both delay and enhance apoptosis in the same cell, suggesting that integrins can play a dual role in the apoptotic progression of leukocytes.

A novel glutathione containing eicosanoid (FOG7) chemotactic for human granulocytes.

A biologically active glutathione adduct of the eicosanoid 5-oxo-eicosatetraenoic acid has been observed as a product formed within the murine peritoneal macrophage. This five-oxo glutathione adduct (FOG(7)) was structurally characterized using electrospray tandem mass spectrometry as a 1,4 Michael addition product 5-oxo-7-glutathionyl-8,11,14-eicosatrienoic acid. FOG(7) was found to be highly potent in stimulating eosinophil as well as neutrophil chemotaxis, also capable of initiating actin polymerization, without elevating intracellular free calcium ion concentration within either the eosinophil or polymorphonuclear leukocyte. These biological responses suggest that either FOG(7) activates a subset of receptors mediating the broader biological activity of the parent eicosanoid 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) or that a receptor not activated by 5-oxo-ETE participates in the chemotactic activity of FOG(7). The only other known biologically active glutathione adduct has been leukotriene C(4) (LTC(4)), another eicosanoid that exerts potent effects through the Cys-LT receptor. The biochemical parallel between the formation of LTC(4) and FOG(7) suggests an interesting mechanism by which biologically active eicosanoids derived from electrophilic intermediates may have unique distribution and prolonged efficacy in vivo.

A hierarchical role for classical pathway complement proteins in the clearance of apoptotic cells in vivo.

The strongest susceptibility genes for the development of systemic lupus erythematosus (SLE) in humans are null mutants of classical pathway complement proteins. There is a hierarchy of disease susceptibility and severity according to the position of the missing protein in the activation pathway, with the severest disease associated with C1q deficiency. Here we demonstrate, using novel in vivo models of apoptotic cell clearance during sterile peritonitis, a similar hierarchical role for classical pathway complement proteins in vivo in the clearance of apoptotic cells by macrophages. Our results constitute the first demonstration of an impairment in the phagocytosis of apoptotic cells by macrophages in vivo in a mammalian system. Apoptotic cells are thought to be a major source of the autoantigens of SLE, and impairment of their removal by complement may explain the link between hereditary complement deficiency and the development of SLE.

A receptor for phosphatidylserine-specific clearance of apoptotic cells.

cytosis 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.

Regulation of phospholipid scramblase activity during apoptosis and cell activation by protein kinase Cdelta.

Phospholipid scramblase induces nonspecific bidirectional movement of phospholipids across the membrane during cell activation and has been proposed to mediate the appearance of phosphatidylserine (PS) in the plasma membrane outer leaflet during apoptosis, a cell surface change that is critical for apoptotic cell removal. We report here that protein kinase C (PKC) delta plays an important role in activated transbilayer movement of phospholipids and surface PS exposure by directly enhancing the activity of phospholipid scramblase. Specific inhibition of PKCdelta by rottlerin prevented both apoptosis- and activation-induced scramblase activity. PKCdelta was either selectively cleaved and activated in a caspase 3-dependent manner (during apoptosis) or translocated to the plasma membrane (in stimulated cells) and could directly phosphorylate scramblase immunoprecipitated from Jurkat cells. Furthermore, reconstitution of PKCdelta and scramblase, but not scramblase or PKCdelta alone in Chinese hamster ovary cells demonstrated enhanced scramblase activity.

Role of p38 mitogen-activated protein kinase in a murine model of pulmonary inflammation.

Early inflammatory events include cytokine release, activation, and rapid accumulation of neutrophils, with subsequent recruitment of mononuclear cells. The p38 mitogen-activated protein kinase (MAPK) intracellular signaling pathway plays a central role in regulating a wide range of inflammatory responses in many different cells. A murine model of mild LPS-induced lung inflammation was developed to investigate the role of the p38 MAPK pathway in the initiation of pulmonary inflammation. A novel p38 MAPK inhibitor, M39, was used to determine the functional consequences of p38 MAPK activation. In vitro exposure to M39 inhibited p38 MAPK activity in LPS-stimulated murine and human neutrophils and macrophages, blocked TNF-alpha and macrophage inflammatory protein-2 (MIP-2) release, and eliminated migration of murine neutrophils toward the chemokines MIP-2 and KC. In contrast, alveolar macrophages required a 1000-fold greater concentration of M39 to block release of TNF-alpha and MIP-2. Systemic inhibition of p38 MAPK resulted in significant decreases in the release of TNF-alpha and neutrophil accumulation in the airspaces following intratracheal administration of LPS. Recovery of MIP-2 and KC from the airspaces was not affected by inhibition of p38 MAPK, and accumulation of mononuclear cells was not significantly reduced. When KC was instilled as a proinflammatory stimulus, neutrophil accumulation was significantly decreased by p38 MAPK inhibition independent of TNF-alpha or LPS. Together, these results demonstrate a much greater dependence on the p38 MAPK cascade in the neutrophil when compared with other leukocytes, and suggest a means of selectively studying and potentially modulating early inflammation in the lung.

Transcriptional and translational regulation of inflammatory mediator production by endogenous TGF-beta in macrophages that have ingested apoptotic cells.

We recently reported that phagocytosis of apoptotic cells inhibits the release of inflammatory cytokines by human macrophages. In this paper we show that apoptotic cell uptake by mouse J774 macrophages also inhibits the synthesis and secretion of the chemokines, macrophage inflammatory protein-2 (Mip-2), KC, and Mip-1alpha (but not that of monocyte chemoattractant protein-1 (MCP-1)/JE), and increases TGF-beta formation. Anti-TGF-beta neutralizing Abs largely reversed the inhibitory effect of apoptotic cell uptake, and accordingly, exogenous TGF-beta down-regulated the synthesis of the same mediators. Apoptotic cell ingestion or TGF-beta also inhibited Mip-2 and Mip-1alpha gene expression in LPS-treated J774 cells, whereas TNF-alpha mRNA levels were unaffected. Importantly, TGF-beta pretreatment of J774 cells did not significantly alter chemokine and TNF mRNA stability. Finally, we found that apoptotic cell uptake and TGF-beta did not modulate NF-kappaB or AP-1 DNA binding in J774 cells. We conclude that the decreased production of chemokines and TNF resulting from apoptotic cell ingestion is largely mediated by a common event, i.e., feedback inhibition by endogenous TGF-beta, but involves different mechanisms. Whereas TNF-alpha production appears to be translationally down-regulated, the suppression of most chemokines investigated appears to reflect transcriptional inhibition. In a broader context, the impairment of chemokine and TNF generation by apoptotic cell uptake might represent an important mechanism contributing to the resolution of inflammation. An additional consequence could be the selective recruitment of monocytes into inflammatory sites, as MCP-1/JE production by mouse macrophages was unaffected by apoptotic cell uptake, in contrast to other chemokines.