Phagocytosis of apoptotic cells and the resolution of inflammation

Clearance of apoptotic cells by phagocytic cells plays a significant role in the resolution of inflammation, protecting tissue from harmful exposure to the inflammatory and immunogenic contents of dying cells. Apoptosis induces cell surface changes that are important for recognition and engulfment of cells by phagocytes. These changes include alterations in surface sugars, externalization of phosphatidylserine and qualitative changes in the adhesion molecule ICAM-3. Several studies have contributed to clarify the role of the receptors on the surface of phagocytes that are involved in apoptotic cell clearance. The phagocytic removal of apoptotic cells does not elicit pro-inflammatory responses; in contrast, apoptotic cell engulfment appears to activate signals that suppress release of pro-inflammatory cytokines. Therefore, clearance of apoptotic leucocytes is implicated in the resolution of inflammation and mounting evidence suggests that defective clearance of apoptotic cells contributes to inflammatory and autoimmune diseases. Defining the ligands on apoptotic cells and the corresponding receptors on phagocytes with which they engage, is likely to lead to the development of novel anti-inflammatory pro-resolution drugs. In this article, we will review the recognition and signaling mechanisms involved in the phagocytosis of apoptotic cells as well as the role of endogenous compounds that play a relevant role in the modulation of inflammation. We will also discuss what is currently known about diseases that may reflect impaired phagocytosis and the consequences on inflammation and immune responses.     

Modulation of FcR function by complement: subcomponent C1q enhances the phagocytosis of IgG-opsonized targets by human monocytes and culture-derived macrophages

We have investigated the interaction of C1q, a subunit of the first component of complement, with human monocytes and culture-derived macrophages. Adherence of these mononuclear phagocytes to surfaces coated with C1q induced a marked enhancement of the phagocytosis of sheep erythrocytes opsonized with IgG anti-Forssman antibody (EA-IgG). This C1q-mediated enhancement of phagocytosis was dose dependent, and was specifically blocked by pretreatment of the C1q-coated surfaces with F(ab')2 anti-C1q. The augmentation of FcR-mediated phagocytosis by C1q was determined to be a result of the interaction between the C1q and the phagocytic effector cell, and was not due to interaction between the surface-bound C1q and the EA-IgG. Neither resting nor N-formyl-methionyl-leucyl-phenylalanine-stimulated polymorphonuclear leukocytes were induced by C1q to increase FcR-mediated phagocytosis. Experiments conducted with purified fragments of C1q suggest that the C1q phagocytosis enhancement signal resides in the collagen-like tail domain of the molecule. This region is the same portion of the molecule previously shown to interact with the cell surface C1q receptor. Native type I collagen was unable to enhance FcR-mediated phagocytosis by mononuclear phagocytes. It has been demonstrated that C1q can be localized to areas of inflammation, and additionally C1q can be secreted by macrophages in culture. In view of these findings and the results of our present study, we hypothesize that C1q could provide local, direct, and non-opsonic enhancement of phagocytosis by mononuclear phagocytes in areas of infection and inflammation.     

Requirement for Rho GTPases and PI 3-kinases during apoptotic cell phagocytosis by macrophages

In vivo, apoptotic cells are removed by surrounding phagocytes, a process thought to be essential for tissue remodeling and the resolution of inflammation [1]. Although apoptotic cells are known to be efficiently phagocytosed by macrophages, the mechanisms whereby their interaction with the phagocytes triggers their engulfment have not been described in mammals. Here, we report that primary murine bone marrow-derived macrophages (using alpha(v)beta(3) integrin for apoptotic cell uptake) extend lamellipodia to engulf apoptotic cells and form an actin cup where phosphotyrosine accumulates. Rho GTPases and PI 3-kinases have been widely implicated in the regulation of the actin cytoskeleton [2, 3]. We show that inhibition of Rho GTPases by Clostridium difficile toxin B prevents apoptotic cell phagocytosis and inhibits the accumulation of both F-actin and phosphotyrosine. Importantly, the Rho GTPases Rac1 and Cdc42 are required for apoptotic cell uptake whereas Rho inhibition enhances uptake. The PI 3-kinase inhibitor LY294002 also prevents apoptotic cell phagocytosis but has no effect on the accumulation of F actin and phosphotyrosine. These results indicate that both Rho GTPases and PI 3-kinases are involved in apoptotic cell phagocytosis but that they play distinct roles in this process.     

Evolutionary Conservation of Divergent Pro-Inflammatory and Homeostatic Responses in Lamprey Phagocytes

In higher vertebrates, phagocytosis plays a critical role in development and immunity, based on the internalization and removal of apoptotic cells and invading pathogens, respectively. Previous studies describe the effective uptake of these particles by lower vertebrate and invertebrate phagocytes, and identify important molecular players that contribute to this internalization. However, it remains unclear if individual phagocytes mediate internalization processes in these ancient organisms, and how this impacts the balance of pro-inflammatory and homeostatic events within their infection sites. Herein we show that individual phagocytes of the jawless vertebrate Petromyzon marinus (sea lamprey), like those of teleost fish and mice, display the capacity for divergent pro-inflammatory and homeostatic responses following internalization of zymosan and apoptotic cells, respectively. Professional phagocytes (macrophages, monocytes, neutrophils) were the primary contributors to the internalization of pro-inflammatory particles among goldfish (C. auratus) and lamprey (P. marinus) hematopoietic leukocytes. However, goldfish showed a greater ability for zymosan phagocytosis when compared to their jawless counterparts. Coupled to this increase was a significantly lower sensitivity of goldfish phagocytes to homeostatic signals derived from apoptotic cell internalization. Together, this translated into a significantly greater capacity for induction of antimicrobial respiratory burst responses compared to lamprey phagocytes, but also a decreased efficacy in apoptotic cell-driven leukocyte homeostatic mechanisms that attenuate this pro-inflammatory process. Overall, our results show the long-standing evolutionary contribution of intrinsic phagocyte mechanisms for the control of inflammation, and illustrate one effective evolutionary strategy for increased responsiveness against invading pathogens. In addition, they highlight the need for development of complementary regulatory mechanisms of inflammation to ensure continued maintenance of host integrity amidst increasing challenges from invading pathogens.     

A chimerical phagocytosis model reveals the recruitment by Sertoli cells of autophagy for the degradation of ingested illegitimate substrates

Phagocytosis and autophagy are typically dedicated to degradation of substrates of extrinsic and intrinsic origins respectively. Although overlaps between phagocytosis and autophagy were reported, the use of autophagy for ingested substrate degradation by nonprofessional phagocytes has not been described. Blood-separated tissues use their tissue-specific nonprofessional phagocytes for homeostatic phagocytosis. In the testis, Sertoli cells phagocytose spermatid residual bodies produced during germ cell differentiation. In the retina, pigmented epithelium phagocytoses shed photoreceptor tips produced during photoreceptor renewal. Spermatid residual bodies and shed photoreceptor tips are phosphatidylserine-exposing substrates. Activation of the tyrosine kinase receptor MERTK, which is implicated in phagocytosis of phosphatidylserine-exposing substrates, is a common feature of Sertoli and retinal pigmented epithelial cell phagocytosis. The major aim of our study was to investigate to what extent phagocytosis by Sertoli cells may be tissue specific. We analyzed in Sertoli cell cultures that were exposed to either spermatid residual bodies (legitimate substrates) or retina photoreceptor outer segments (illegitimate substrates) the course of the main phagocytosis stages. We show that whereas substrate binding and ingestion stages occur similarly for legitimate or illegitimate substrates, the degradation of illegitimate but not of legitimate substrates triggers autophagy as evidenced by the formation of double-membrane wrapping, MAP1LC3A-II/LC3-II clustering, SQSTM1/p62 degradation, and by marked changes in ATG5, ATG9 and BECN1/Beclin 1 protein expression profiles. The recruitment by nonprofessional phagocytes of autophagy for the degradation of ingested cell-derived substrates is a novel feature that may be of major importance for fundamentals of both apoptotic substrate clearance and tissue homeostasis.     

Phagocytosis of apoptotic cells

Removal of apoptotic cells by phagocytes plays an important role in many biological processes, including embryological development and tissue remodelling. In addition, it has become apparent that one of the key mechanisms for the successful resolution of inflammation is the orchestrated clearance of apoptotic inflammatory cells by phagocytes (e.g., macrophages and dendritic cells) and other cells known to have phagocytic capacity (e.g., hepatocytes, endothelial cells, epithelial cells, etc.). Furthermore, phagocytosis of apoptotic cells is an active and highly regulated process that not only serves to remove potentially histotoxic cells from the inflammatory milieu, but also directs the phenotype of the phagocytic cell to be anti-inflammatory. Convincing evidence has been presented that reduced or dysregulated phagocytosis of apoptotic cells contributes to the development and propagation of inflammatory disorders. Conversely, enhanced phagocytosis of apoptotic cells may be exploited for therapeutic gain. Indeed, powerful anti-inflammatory drugs such as the glucocorticoids have been shown to augment clearance of apoptotic cells which may contribute to their therapeutic effectiveness. In this chapter, we describe methods for studying phagocytosis of apoptotic cells.     

Beta-2-glycoprotein 1-dependent macrophage uptake of apoptotic cells. Binding to lipoprotein receptor-related protein receptor family members

The recognition and removal of apoptotic cells is critical to development, tissue homeostasis, and the resolution of inflammation. Many studies have shown that phagocytosis is regulated by signaling mechanisms that involve distinct ligand-receptor interactions that drive the engulfment of apoptotic cells. Studies from our laboratory have shown that the plasma protein beta-2-glycoprotein 1 (beta2GP1), a member of the short consensus repeat superfamily, binds phosphatidylserine-containing vesicles and apoptotic cells and promotes their bridging and subsequent engulfment by phagocytes. The phagocyte receptor for the protein/apoptotic cell complex, however, is unknown. Here we report that a member of the low density lipoprotein receptor-related protein family on phagocytes binds and facilitates engulfment of beta2GP1-phosphatidylserine and beta2GP1-apoptotic cell complexes. Using recombinant beta2GP1, we also show that beta2GP1-dependent uptake is mediated by bridging of the target cell to the phagocyte through the protein C- and N-terminal domains, respectively.     

Turning on the respiratory burst

The respiratory burst is a distinguishing property of phagocytes. It is induced by chemotactic stimulation or phagocytosis and reflects the activation of a membrane-bound enzyme system that transfers electrons from cytosolic NADPH to extracellular oxygen, producing superoxide. The products of the burst are essential for the killing of microorganisms, but are also a cause of tissue damage and inflammation. Studies aimed at a better understanding of the regulation of the respiratory burst should help in the search for new ways to treat infections and inflammation.     

Phagocytosis

Phagocytosis is the process of recognition and engulfment of microorganisms or tissue debris that accumulate during infection, inflammation or wound repair. This ingestion, which is performed most efficiently by migrating, bone marrow-derived cells called ‘professional phagocytes’, is essential for successful host defense. Ingestion results when an invading microorganism is recognized by specific receptors on the phagocyte surface and requires multiple, successive interactions between the phagocyte and the target. Each of these interactions results in a signal transduction event, which is confined to the membrane and cytoskeleton around the ligated receptor and which is required for successful phagocytosis. Many molecules found at sites of inflammation or infection stimulate phagocytosis, so that efficient ingestion is confined to the site of infection or inflammation, which in turn limits the proinflammatory and tissue-destructive processes that accompany phagocytosis. This review summarizes current understanding of this critical component of host defense and of its regulation.     

Neuronal Hyperactivity Disturbs ATP Microgradients,Impairs Microglial Motility,and Reduces Phagocytic Receptor Expression Triggering Apoptosis/Microglial Phagocytosis Uncoupling

Phagocytosis is essential to maintain tissue homeostasis in a large number of inflammatory and autoimmune diseases, but its role in the diseased brain is poorly explored. Recent findings suggest that in the adult hippocampal neurogenic niche, where the excess of newborn cells undergo apoptosis in physiological conditions, phagocytosis is efficiently executed by surveillant, ramified microglia. To test whether microglia are efficient phagocytes in the diseased brain as well, we confronted them with a series of apoptotic challenges and discovered a generalized response. When challenged with excitotoxicity in vitro (via the glutamate agonist NMDA) or inflammation in vivo (via systemic administration of bacterial lipopolysaccharides or by omega 3 fatty acid deficient diets), microglia resorted to different strategies to boost their phagocytic efficiency and compensate for the increased number of apoptotic cells, thus maintaining phagocytosis and apoptosis tightly coupled. Unexpectedly, this coupling was chronically lost in a mouse model of mesial temporal lobe epilepsy (MTLE) as well as in hippocampal tissue resected from individuals with MTLE, a major neurological disorder characterized by seizures, excitotoxicity, and inflammation. Importantly, the loss of phagocytosis/apoptosis coupling correlated with the expression of microglial proinflammatory, epileptogenic cytokines, suggesting its contribution to the pathophysiology of epilepsy. The phagocytic blockade resulted from reduced microglial surveillance and apoptotic cell recognition receptor expression and was not directly mediated by signaling through microglial glutamate receptors. Instead, it was related to the disruption of local ATP microgradients caused by the hyperactivity of the hippocampal network, at least in the acute phase of epilepsy. Finally, the uncoupling led to an accumulation of apoptotic newborn cells in the neurogenic niche that was due not to decreased survival but to delayed cell clearance after seizures. These results demonstrate that the efficiency of microglial phagocytosis critically affects the dynamics of apoptosis and urge to routinely assess the microglial phagocytic efficiency in neurodegenerative disorders.     

Receptor for advanced glycation end products binds to phosphatidylserine and assists in the clearance of apoptotic cells

Clearance of apoptotic cells is necessary for tissue development, homeostasis and resolution of inflammation. The uptake of apoptotic cells is initiated by an 'eat-me' signal, such as phosphatidylserine, on the cell surface and phagocytes recognize the signal by using specific receptors. In this study, we show that the soluble form of the receptor for advanced glycation end products (RAGE) binds to phosphatidylserine as well as to the apoptotic thymocytes. RAGE-deficient (Rage(-/-)) alveolar macrophages showed impaired phagocytosis of apoptotic thymocytes and defective clearance of apoptotic neutrophils in Rage(-/-) mice. Our results indicate that RAGE functions as a phosphatidylserine receptor and assists in the clearance of apoptotic cells.     

芍药苷通过IL-10-STAT3信号通路抑制脂多糖诱导BV2细胞炎症与吞噬作用

小胶质细胞是中枢神经系统中重要免疫细胞,也是炎症反应中的主要效应细胞。芍药苷被证实能有效抑制炎症反应,在调节免疫方面具有巨大药用价值。本文旨在阐明BV2细胞炎症反应中芍药苷对细胞炎症及吞噬的抑制作用,并探索其中潜在机制。体外实验利用脂多糖（lipopolysaccharide,LPS）诱导BV2细胞发生炎症反应,芍药苷能有效抑制BV2细胞TNF-α和NO的产生以及BV2细胞异常增加的吞噬功能,并且在此过程中IL-10-STAT3信号通路被激活;芍药苷的抑制作用在我们使用STAT3抑制剂JSI-124后显著降低,TNF-α和NO的表达量增加、BV2细胞的吞噬功能增强。上述结果表明,芍药苷能有效抑制BV2细胞炎症作用及吞噬作用,这一过程中依赖IL-10-STAT3信号通路的激活。这将加深我们对芍药苷抑制小胶质细胞炎症作用机制的认识。     

Adenosine receptors are expressed during differentiation of monocytes to macrophages in vitro. Implications for regulation of phagocytosis

Ingestion by phagocytes is known to be markedly enhanced by physiologic signals such as cytokines and extracellular matrix proteins which may be found in inflammatory sites. Little investigation has been made of mechanisms that may depress this increased rate of phagocytosis during resolution of inflammation. We show that adenosine can act as an inhibitor of phagocytosis by macrophages derived from in vitro culture of human peripheral blood monocytes. Adenosine (Ado) is equally effective at inhibiting IgG Fc and complement-mediated phagocytosis. However, Ado has no effect on phagocytosis by freshly isolated monocytes. Inhibition by Ado begins after 2 days in culture and reaches a plateau by 5 days; these kinetics of induction of inhibition of phagocytosis parallel an increase in specific Ado binding to the macrophage plasma membrane. Ado binds to cultured monocytes with a Kd of 6 microM. This affinity and the observation that 2-chloroadenosine and 5'-N-ethylcarboxamidadenosine are the most potent inhibitors of phagocytosis suggest that the Ado receptors expressed during monocyte differentiation are of the A2 type. The inhibition of phagocytosis may be mediated by cAMP, a second messenger coupled to A2 receptors in several cell types. Thus, plasma membrane expression of A2 receptors dramatically increases during monocyte differentiation in vitro. These data show that a potentially physiologic mediator can have very different effects on the function of monocytes and macrophages. This suggests a mechanism whereby phagocytic function at inflammatory sites can be down-regulated if and only if signals for the recruitment of new phagocytes have subsided.     

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.     

Identification of calreticulin as a marker for phagocytosis of apoptotic cells in Drosophila

Apoptotic cell phagocytosis is initiated through the specific interaction between markers for phagocytosis present at the surface of targets and their receptors of phagocytes. Although many molecules have been proposed to be phagocytosis markers and receptors in mammals, information as to the identity of those molecules is limited for invertebrate animals. Calreticulin, a molecular chaperone that functions in the lumen of the endoplasmic reticulum, was recently reported to be the second general marker, the membrane phospholipid phosphatidylserine being the first, for mammalian apoptotic cells to be recognized by phagocytes. We here asked whether or not calreticulin serves as a marker for phagocytosis in Drosophila. Phagocytosis of apoptotic S2 cells by Drosophila hemocyte-derived l(2)mbn cells, which we previously showed to occur independent of phosphatidylserine, was inhibited by the addition of anti-calreticulin antibody. This inhibition was observed when the target cells, but not phagocytes, were pre-incubated with the antibody. In addition, RNA interference-mediated reduction of calreticulin expression in apoptotic S2 cells, but not in l(2)mbn cells, reduced the level of phagocytosis. An immunocytochemical analysis revealed that calreticulin is widely distributed at the surface of viable S2 cells. After the induction of apoptosis, cell surface calreticulin seemed to form aggregates, with no change in its amount. Furthermore, in embryos of a mutant Drosophila strain that expresses calreticulin at a reduced level, the level of phagocytosis of apoptotic cells was about a half of that observed in embryos of a wild-type strain. These results collectively indicate that calreticulin is the first molecule to be identified as a marker for phagocytosis of apoptotic cells by Drosophila phagocytes.     

C反应蛋白与动脉粥样硬化

人类C反应蛋白 (C reactiveprotein ,CRP)是在感染和组织损伤时血浆浓度快速、急剧升高的主要的急性期蛋白。CRP可以激活补体和加强吞噬细胞的吞噬而起调理作用 ,从而清除入侵机体的病原微生物和损伤、坏死、凋亡的组织细胞 ,在机体的天然免疫过程中发挥重要的保护作用。关于CRP的研究已经有 70多年的历史 ,传统观点认为CRP是一种非特异的炎症标志物 ,但近十年的研究揭示了CRP直接参与了炎症与动脉粥样硬化等心血管疾病 ,并且是心血管疾病最强有力的预示因子与危险因子     

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare ''professional'' phagocytes - macrophages and dendritic cells to ''non-professional'' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: ''professional'' macrophages and ''semi-professional'' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.     

Development of an in vitro model for the multi-parametric quantification of the cellular interactions between Candida yeasts and phagocytes

We developed a new in vitro model for a multi-parameter characterization of the time course interaction of Candida fungal cells with J774 murine macrophages and human neutrophils, based on the use of combined microscopy, fluorometry, flow cytometry and viability assays. Using fluorochromes specific to phagocytes and yeasts, we could accurately quantify various parameters simultaneously in a single infection experiment: at the individual cell level, we measured the association of phagocytes to fungal cells and phagocyte survival, and monitored in parallel the overall phagocytosis process by measuring the part of ingested fungal cells among the total fungal biomass that changed over time. Candida albicans, C. glabrata, and C. lusitaniae were used as a proof of concept: they exhibited species-specific differences in their association rate with phagocytes. The fungal biomass uptaken by the phagocytes differed significantly according to the Candida species. The measure of the survival of fungal and immune cells during the interaction showed that C. albicans was the more aggressive yeast in vitro, destroying the vast majority of the phagocytes within five hours. All three species of Candida were able to survive and to escape macrophage phagocytosis either by the intraphagocytic yeast-to-hyphae transition (C. albicans) and the fungal cell multiplication until phagocytes burst (C. glabrata, C. lusitaniae), or by the avoidance of phagocytosis (C. lusitaniae). We demonstrated that our model was sensitive enough to quantify small variations of the parameters of the interaction. The method has been conceived to be amenable to the high-throughput screening of mutants in order to unravel the molecular mechanisms involved in the interaction between yeasts and host phagocytes.     

Cutting edge: the Wiskott-Aldrich syndrome protein is required for efficient phagocytosis of apoptotic cells

Phagocytosis of apoptotic cells by macrophages and dendritic cells is necessary for clearance of proinflammatory debris and for presentation of viral, tumor, and self Ags. While a number of receptors involved in the cognate recognition of apoptotic cells by phagocytes have been identified, the signaling events that result in internalization remain poorly understood. Here we demonstrate that clearance of apoptotic cells is accompanied by recruitment of the Wiskott-Aldrich syndrome (WAS) protein to the phagocytic cup and that it's absence results in delayed phagocytosis both in vitro and in vivo. Therefore, we propose that WAS protein plays an important and nonredundant role in the safe removal of apoptotic cells and that deficiency contributes significantly to the immune dysregulation of WAS. The efficiency of apoptotic cell clearance may be a key determinant in the suppression of tissue inflammation and prevention of autoimmunity.     

Intervertebral disc cells as competent phagocytes in vitro: implications for cell death in disc degeneration

Introduction

Apoptosis has been reported to occur in the intervertebral disc. Elsewhere in the body, apoptotic cells are cleared from the system via phagocytosis by committed phagocytes such as macrophages, reducing the chance of subsequent inflammation. These cells, however, are not normally present in the disc. We investigated whether disc cells themselves can be induced to become phagocytic and so have the ability to ingest and remove apoptotic disc cells, minimising the damage to their environment.

Method

Bovine nucleus pulposus cells from caudal intervertebral discs were grown in culture and exposed to both latex particles (which are ingested by committed phagocytes) and apoptotic cells. Their response was monitored via microscopy, including both fluorescent and video microscopy, and compared with that seen by cell lines of monocytes/macrophages (THP-1 and J774 cells), considered to be committed phagocytes, in addition to a nonmacrophage cell line (L929 fibroblasts). Immunostaining for the monocyte/macrophage marker, CD68, was also carried out.

Results

Disc cells were able to ingest latex beads at least as efficiently, if not more so, than phagocytic THP-1 and J774 cells. Disc cells ingested a greater number of beads per cell than the committed phagocytes in a similar time scale. In addition, disc cells were able to ingest apoptotic cells when cocultured in monolayer with a UV-treated population of HeLa cells. Apoptotic disc cells, in turn, were able to stimulate phagocytosis by the committed macrophages. CD68 immunostaining was strong for THP-1 cells but negligible for disc cells, even those that had ingested beads.

Conclusion

In this study, we have shown that intervertebral disc cells are capable of behaving as competent phagocytes (that is, ingesting latex beads) and apoptotic cells. In terms of number of particles, they ingest more than the monocyte/macrophage cells, possibly due to their greater size. The fact that disc cells clearly can undergo phagocytosis has implications for the intervertebral disc in vivo. Here, where cell death is reported to be common yet there is normally no easy access to a macrophage population, the endogenous disc cells may be encouraged to undergo phagocytosis (for example, of neighbouring cells within cell clusters).