Amateur Phagocytes Research Articles

Unexpected Role of Nonimmune Cells: Amateur Phagocytes.

Effective and efficient efferocytosis of dead cells and associated cellular debris are critical to tissue homeostasis and healing of injured tissues. This important task was previously thought to be restricted to professional phagocytes (PPs). However, accumulating evidence has revealed another type of phagocyte, the amateur phagocyte (AP), which can also participate in efferocytosis. APs are non-myeloid progenitor/nonimmune cells that include differentiated cells (e.g., epithelial cells, fibroblasts, and endothelial cells [ECs]) and stem cells (e.g., neuronal progenitor cells and mesenchymal cells) and can be found throughout the human body. Studies have shown that APs have two prominent roles: identifying and removing dead cells presumably before PPs reach the site of injury and assisting PPs in the removal of cell corpses and the resolution of inflamed tissue. With respect to the engulfment and degradation of dead cells, APs are slower and less efficient than PPs. However, APs are fundamental to preventing the spread of inflammation over a large area. In this review, we present the diversity and characteristics of healthy and non-neoplastic APs in mammals. We also propose a hypothetical mechanism of the efferocytosis of immunoglobulin G (IgG)-opsonized myelin debris by ECs (APs). Furthermore, the ingestion and clearance of dead cells can induce proinflammatory or anti-inflammatory cytokine production, endothelial activation, and cellular fate transition, which contribute to the progression of disease. An understanding of the role of APs is necessary to develop effective intervention strategies, including potential molecular targets for clinical diagnosis and drug development, for inflammation-related diseases.

Amateur phagocytes.

Amateur phagocytes.

Elevated Serum Lysophosphatidylcholine in Patients with Systemic Lupus Erythematosus Impairs Phagocytosis of Necrotic Cells In Vitro.

Impaired clearance of dying and dead cells by professional and amateur phagocytes plays a crucial role in the etiology of systemic lupus erythematosus (SLE). While dying, cells expose and release a plethora of eat-me and find-me signals to ensure their timely removal before entering the dangerous stage of secondary necrosis. A well-described chemoattractant for macrophages is dying cell-derived lysophosphatidylcholine (LPC). However, its implications for and/or its association with SLE disease, so far, have not been examined. In the present study, we analyzed the LPC serum concentrations of patients with SLE and rheumatoid arthritis (RA). Subsequently, we examined if and to which extent the measured serum concentrations of LPC and an LPC-rich environment can impact the phagocytosis of necrotic cells. Sera from patients with SLE, RA, and normal healthy donors (NHD) were characterized for several parameters, including LPC concentrations. Phagocytosis of dead cells by human macrophages in the presence of SLE and NHD sera was quantified. Additionally, the impact of exogenously added, purified LPC on phagocytosis was analyzed. Patients with SLE had significantly increased LPC serum levels, and high serum LPC of SLE patients correlated significantly with impaired phagocytosis of dead cells in the presence of heat-inactivated serum. Phagocytosis in the presence of sera from NHD showed no correlation to LPC levels, but exogenous addition of purified LPC in the range as measured in SLE patients' sera led to a concentration-dependent decrease. Our data show that high levels of LPC as observed in the sera of SLE patients have a negative impact on the clearance of dead cells by macrophages. Chemoattraction requires a concentration gradient. The higher the LPC concentration surrounding a dying or dead cell, the smaller the achievable gradient upon LPC release will be. Thus, it is feasible to assume that elevated LPC levels can interfere with the build-up of a local LPC gradient during cell death, and hence might play a role in the establishment and/or perpetuation of SLE disease.

Apoptotic Cell Clearance in Drosophila melanogaster.

The swift clearance of apoptotic cells (ACs) (efferocytosis) by phagocytes is a critical event during development of all multicellular organisms. It is achieved through phagocytosis by professional or amateur phagocytes. Failure in this process can lead to the development of inflammatory autoimmune or neurodegenerative diseases. AC clearance has been conserved throughout evolution, although many details in its mechanisms remain to be explored. It has been studied in the context of mammalian macrophages, and in the nematode Caenorhabditis elegans, which lacks “professional” phagocytes such as macrophages, but in which other cell types can engulf apoptotic corpses. In Drosophila melanogaster, ACs are engulfed by macrophages, glial, and epithelial cells. Drosophila macrophages perform similar functions to those of mammalian macrophages. They are professional phagocytes that participate in phagocytosis of ACs and pathogens. Study of AC clearance in Drosophila has identified some key elements, like the receptors Croquemort and Draper, promoting Drosophila as a suitable model to genetically dissect this process. In this review, we survey recent works of AC clearance pathways in Drosophila, and discuss the physiological outcomes and consequences of this process.

The Pallbearer E3 Ligase Promotes Actin Remodeling via RAC in Efferocytosis by Degrading the Ribosomal Protein S6

Clearance of apoptotic cells (efferocytosis) is achieved through phagocytosis by professional or amateur phagocytes. It is critical for tissue homeostasis and remodeling in all animals. Failure in this process can contribute to the development of inflammatory autoimmune or neurodegenerative diseases. We found previously that the PALL-SCF E3-ubiquitin ligase complex promotes apoptotic cell clearance, but it remained unclear how it did so. Here we show that the F-box protein PALL interacts with phosphorylated ribosomal protein S6 (RpS6) to promote its ubiquitylation and proteasomal degradation. This leads to RAC2 GTPase upregulation and activation and F-actin remodeling that promotes efferocytosis. We further show that the specific role of PALL in efferocytosis is driven by its apoptotic cell-induced nuclear export. Finding a role for RpS6 in the negative regulation of efferocytosis provides the opportunity to develop new strategies to regulate this process.

Apoptosis in Living Animals Is Assisted by Scavenger Cells and Thus May Not Mainly Go through the Cytochrome C-Caspase Pathway

Because billions of cells die every day in their bodies, animals have evolutionarily developed apoptosis to preserve the tissue environment from adverse effects of dead cells, a process achieved via phagocytosis of the cell corpses by professional or amateur phagocytes that are collectively referred to as scavengers. Hence, apoptosis is a merger of two procedures separately occurring inside the dying and the scavenger cells, respectively. The task of apoptosis research is to study how these death procedures occur without hurting the host tissues, and recruitment of in vitro system into the study must be justified for this purpose. Cells in culture have no motivation to preserve the environment, and their death does not involve corpse clearance by scavengers. Therefore, programmed cell death in culture should be redefined, for example as stress-induced cell death, to avoid many sources of confusions, since the word “apoptosis” had already been defined, prior to the era of cell culture, as a silent and beneficial cell suicide with corpse clearance as a distinctive hallmark. We should start over again on apoptosis research by determining whether different physiological apoptotic procedures in animals involve the cytochrome c-caspase pathway, since it has been established from cultured cells as a central mechanism of “apoptosis” but whether it overarches any physiological apoptotic procedure in animals is still unclear. Probably, cells in living animals are programmed to use scavengers to assist their apoptosis but cells in culture have no scavengers to help and thus need to go mainly through the cytochrome c-caspase pathway.

Fluorescent Probes Detecting the Phagocytic Phase of Apoptosis: Enzyme-Substrate Complexes of Topoisomerase and DNA

In apoptosis, the initial self-driven suicide phase generates cellular corpses which are digested in the phagolysosomes of professional and amateur phagocytes during the subsequent waste-management phase. This ensures the complete elimination of the genetic material which often contains pathological, viral or cancerous DNA sequences. Although the phagocytic phase is critical for the efficient execution of apoptosis, there are currently few methods specifically adapted for its detailed visualization in the fixed tissue section format. To resolve this we developed new fluorescent probes for in situ research. The probes selectively visualize active phagocytic cells of any lineage (professional, amateur phagocytes or surrounding tissue cells) which engulf and digest apoptotic cell DNA. These fluorescent probes are the covalently-bound enzyme-DNA intermediates produced in a topoisomerase reaction with specific “starting” oligonucleotides. They detect a specific marker of DNase II cleavage activity, which occurs exclusively in phagolysosomes of the cells that engulfed apoptotic nuclei. The probes provide snap-shot images of the digestion process occurring in cellular organelles responsible for the actual execution of phagocytic degradation of apoptotic cell corpses. We applied the probes for visualization of the phagocytic reaction in tissue sections of normal thymus and in several human lymphomas. We also discuss the nature, stability and properties of DNase II-type breaks as a marker of phagocytic activity. This development provides a useful fluorescent tool for studies of pathologies where clearance of dying cells is essential, such as cancers, inflammation, infection and auto-immune disorders.

Differentiation of the Mammary Epithelial Cell during Involution: Implications for Breast Cancer

That milk secretion is not the final differentiated state of the mammary alveolar cells is a relatively new concept. Recent work has suggested that secreting, mammary epithelial cells (MECs) have another function to perform before they undergo cell death in the involuting mammary gland. That is, they help in the final clearance and breakdown of their neighboring cells (and likely residual milk as well.) They become, for a short time, amateur phagocytes, or efferocytes, and then are believed to die and be cleared themselves. Although relatively little study has been made of this change in the functional state of the MEC, nevertheless we may speculate from the involution literature, and extend findings from other systems of apoptotic cell clearance, on some of the mechanisms involved. And with the finding that involution may represent a unique susceptibility window for the progression of metastatic breast cancer, we may suggest areas for future research along these lines as well.

The lonely death: Chondrocyte apoptosis in TNF-induced arthritis

Inflammatory joint disease typically provokes progressive cartilage damage. The proliferative synovial inflammatory tissue directly invades the cartilage and induces the expression and activation of degrading enzymes such as matrix metalloproteases (MMPs) and aggrecanases. However, also chondrocyte apoptosis has been observed in cartilage samples of inflamed joints. It remains unclear whether this is a secondary phenomenon due to cartilage damage or a primary event initiated by the synovial inflammation. To determine the presence or absence of chondrocyte death in experimental arthritis, we longitudinally assessed proteoglycan depletion and chondrocyte apoptosis in paw sections from human tumor necrosis factor transgenic (hTNFtg) mice and wild-type littermates. Whereas, wild-type mice showed no signs of cartilage damage, hTNFtg mice exhibited progressive proteoglycan loss starting at clinical onset of arthritis. However, we already found the first apoptotic chondrocytes well before cartilage matrix breakdown occurred indicating that chondrocyte death can be induced before matrix resorption. Chondrocyte death could constantly be observed until late stages of arthritis causing a continuous increase in the number of empty cartilage lacunae. As apoptotic cells in cartilage cannot be cleared by phagocytes due to their spatial isolation in the avascular lacunae of cartilage, having no contact to professional or amateur phagocytes. The dying cells are compelled to undergo a “lonely death” inevitable ending up in secondary necrosis giving rise to perpetuation of a pro-inflammatory cascade. These data indicate that chondrocyte death may play a primary role in inflammatory arthritis fuelling cartilage inflammation and damage due to secondary necrosis.

Epithelial cells as phagocytes: apoptotic epithelial cells are engulfed by mammary alveolar epithelial cells and repress inflammatory mediator release

Clearance of apoptotic cells is critical to tissue homeostasis and resolution of inflammatory lesions. Macrophages are known to remove dying cells and release anti-inflammatory mediators in response; however, many cells traditionally thought of as poor phagocytes can mediate this function as well. In the lactating mammary gland following weaning, alveolar epithelial cell death is massive, yet the gland involutes rapidly, attaining its prepregnancy state in a matter of days. We found histologic evidence of apoptotic cell phagocytosis by viable mammary epithelial cells (MEC) in the involuting mouse mammary gland. Cultured MEC were able to engulf apoptotic cells in vitro, utilizing many of the same receptors used by macrophages, including the phosphatidylserine receptor (PSR), CD36, the vitronectin receptor alpha(v)beta3, and CD91. In addition, MEC, like macrophages, produced TGFbeta in response to stimulation of the PSR by apoptotic cells or the anti-PSR ab 217G8E9, and downregulated endotoxin-stimulated proinflammatory cytokine production. These data support the hypothesis that amateur phagocytes play a significant role in apoptotic cell clearance and its regulation of inflammation.

Immunopathological consequences of the loss of engulfment genes: the case of ABCA1

Programmed cell death plays a crucial role in the maintenance of cell homeostasis. An initial, effector phase leads to the generation of apoptotic corpses and is closely followed by a swift clearance by professional or amateur phagocytes. Several aspects distinguish this latter process of engulfment of dying cells from the classical forms of phagocytosis. They concern all aspects of the process from the recognition of the prey to the final outcome, i.e. immunological silence. The engulfment of dead cells is a process highly conserved through evolution and it has been studied in parallel in two systems, mammalian cells and the nematode C. elegans. ABCA1 and its ortholog CED-7 in the nematode are key players of engulfment. Their mode of action is somehow original in the panorama of engulfment receptors since they act as lipid transporters. While in the worm the loss of CED-7 has phenotypic consequences exclusively on engulfment, in the mouse the deletion of ABCA1 by homologous recombination has highlighted broad consequences on macrophage biology. Among those we will discuss here the aberrant responses of ABCA1-/- mice to Plasmodium berghei ANKA infection, concerning in particular the development of cerebral malaria (CM), a cytokine-induced immunopathology. This syndrome involves a central role of monocytes and, as shown recently, high levels of circulating microparticles. It was found that ABCA1 loss completely protects against CM and its associated mortality. This observation, together with the demonstration of quantitative and functional modifications of microparticles, suggests that microparticles may be involved in CM pathogenesis. The ABCA1 transporter thus appears to control susceptibility to CM, thereby providing new insights in its pathophysiological mechanisms and potential new therapeutic avenues.

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.