Immune complexes bind preferentially to Fc gamma RIIA (CD32) on apoptotic neutrophils, leading to augmented phagocytosis by macrophages and release of proinflammatory cytokines

Many human inflammatory diseases are associated with tissue deposition of immune complexes and influx of neutrophils. We show that immune complexes bind preferentially to apoptotic neutrophils via FcgammaRIIA (CD32) and that increased binding is associated with clustering of immune complexes on the plasma membrane of the apoptotic cell. Phagocytosis of immune complex-opsonized apoptotic neutrophils by human macrophages was substantially enhanced (4.4-fold increase compared with control apoptotic neutrophils) and stimulated macrophages to release the proinflammatory cytokines TNF-alpha and IL-6. Immune complexes may perturb the normal pathways for clearance of apoptotic neutrophils by augmenting their clearance at the price of proinflammatory cytokine release. This represents a novel mechanism by which immune complexes may modulate the resolution of inflammation.     

Friendly fire against neutrophils: proteolytic enzymes confuse the recognition of apoptotic cells by macrophages

Physiologically the only acceptable fate for almost all damaged or unwanted cells is their apoptotic death, followed by engulfment of the corpses by healthy neighbors or professional phagocytes. Efficient clearance of cells that have succumbed to apoptosis is crucial for normal tissue homeostasis, and for the modulation of immune responses. The disposal of apoptotic cells is finely regulated by a highly redundant system of receptors, bridging molecules and 'eat me' signals. The complexity of the system is reflected by the term: 'engulfment synapse', used to describe the interaction between a phagocytic cell and its target. In healthy humans, dying neutrophils are the most abundant and important targets for such recognition and engulfment. In inflammation the scope and importance of this complicated task is further increased. Paradoxically, despite growing evidence highlighting the priority of neutrophils clearance, the recognition of these cells by phagocytes is not as well understood as the recognition of other apoptotic cell types. New findings indicate that the interaction of phosphatidylserine (PS) on apoptotic neutrophils with its receptor on macrophages is not as critical for the specific clearance of neutrophil corpses it was previously believed. In this review we focus on recent findings regarding alternative, PS-independent "eat me" signals expressed on neutrophils during cell death and activation. Based on our own research, we emphasize the clearance of dying neutrophils, especially at the focus of bacterial infection; and the associated inflammatory reaction, which occurs in a highly proteolytic milieu containing both host and bacteria-derived proteinases. In these environments, eat-me signals expressed by neutrophils are drastically modified; arguing against the phospholipid-based detection of apoptotic cells, but supporting the importance of proteinaceous ligand(s) for the recognition of neutrophils by macrophages. In this context we discuss the effect of the gingipain R (Rgp) proteinases from Porphyromonas gingivalis on neutrophils interactions with macrophages. Since the recognition of apoptotic neutrophils is an important fundamental process, serving multiple functions in the regulation of immunity and homeostasis, we hypothesize that many pathogenic bacteria may have developed similar strategies to confuse macrophage-neutrophil interaction as a common pathogenic strategy.     

Six-microns-under acts upstream of Draper in the glial phagocytosis of apoptotic neurons

The removal of apoptotic cells by phagocytic neighbors is essential for metazoan development but remains poorly characterized. Here we report the discovery of a Drosophila phagocytosis receptor, Six-microns-under (SIMU), which is expressed in highly phagocytic cell types during development and required for efficient apoptotic cell clearance by glia in the nervous system and by macrophages elsewhere. SIMU is part of a conserved family of proteins that includes CED-1 and Draper (DRPR). Phenotypic analysis reveals that simu acts upstream of drpr in the same pathway and affects the recognition and engulfment of apoptotic cells, while drpr affects their subsequent degradation. SIMU strongly binds to apoptotic cells, presumably through its EMILIN-like domain, but requires no membrane anchoring, suggesting that it can function as a bridging molecule. Our study introduces an important factor in tissue-resident apoptotic clearance and underscores the prominent role of glia as "semiprofessional" phagocytes in the nervous system.     

Vascular endothelial growth factor enhances macrophage clearance of apoptotic cells

Efficient clearance of apoptotic cells from the lung by alveolar macrophages is important for the maintenance of tissue structure and function. Lung tissue from humans with emphysema contains increased numbers of apoptotic cells and decreased levels of vascular endothelial growth factor (VEGF). Mice treated with VEGF receptor inhibitors have increased numbers of apoptotic cells and develop emphysema. We hypothesized that VEGF regulates apoptotic cell clearance by alveolar macrophages (AM) via its interaction with VEGF receptor 1 (VEGF R1). Our data show that the uptake of apoptotic cells by murine AMs and human monocyte-derived macrophages is inhibited by depletion of VEGF and that VEGF activates Rac1. Antibody blockade or pharmacological inhibition of VEGF R1 activity also decreased apoptotic cell uptake ex vivo. Conversely, overexpression of VEGF significantly enhanced apoptotic cell uptake by AMs in vivo. These results indicate that VEGF serves a positive regulatory role via its interaction with VEGF R1 to activate Rac1 and enhance AM apoptotic cell clearance.     

Novel role of ICAM3 and LFA-1 in the clearance of apoptotic neutrophils by human macrophages

Apoptotic cells express eat-me signals which are recognized by several receptors mainly on professional phagocytes of the mononuclear phagocyte system. This “engulfment synapse” can define a safe and effective clearance of apoptotic cells in order to maintain tissue homeostasis in the entire body. We show that the expression of four genes related to apoptotic cell clearance is strongly up-regulated in human macrophages 30 min after administration of apoptotic neutrophils. Out of these the significant role of the up-regulated intercellular adhesion molecule 3 (ICAM3) in phagocytosis of apoptotic neutrophils could be demonstrated in macrophages by gene silencing as well as treatment with blocking antibodies. Blocking ICAM3 on the surface of apoptotic neutrophils also resulted in their decreased uptake which confirmed its role as an eat-me signal expressed by apoptotic cells. In macrophages but not in neutrophils silencing and blocking integrin alphaL and beta2 components of lymphocyte function-associated antigen 1 (LFA-1), which can strongly bind ICAM3, resulted in a decreased phagocytosis of apoptotic cells indicating its possible role to recognize ICAM3 on the surface of apoptotic neutrophils. Finally, we report that engulfing portals formed in macrophages during phagocytosis are characterized by accumulation of ICAM3, integrin alphaL and beta2 which show co-localization on the surface of phagocytes. Furthermore, their simultaneous knock-down in macrophages resulted in a marked deficiency in phagocytosis and a slight decrease in the anti-inflammatory effect of apoptotic neutrophils. We propose that ICAM3 and LFA-1 act as recognition receptors in the phagocytosis portals of macrophages for engulfment of apoptotic neutrophils.     

TGF-beta influences the life and death decisions of T lymphocytes

TGF-beta is a powerful mediator of immune cell phenotype and function. In TGF-beta1 homozygous null mice, aberrant regulation of the immune response culminates in lethal cardiopulmonary inflammation. In dissecting the underlying mechanisms leading to the attack of self, a role for TGF-beta1 in controlling apoptosis and T cell selection patterns was uncovered. Increased levels of apoptosis and TCR mediated cell death disrupted normal negative and positive T cell selection in the thymus. Moreover, in peripheral T cell populations, increased T lymphocyte death was associated with increased expression of apoptosis-inducing receptors. Persistent activation of T cells engendered unchecked apoptosis which, rather than reducing, further exacerbated, tissue inflammation due to the absence of TGF-beta1. TGF-beta, normally generated by macrophages during clearance of apoptotic cells contributes to dampening of inflammatory sequelae associated with phagocytosis. Collectively, these data demonstrate a pivotal role for TGF-beta in multiple stages of T cell apoptosis, selection, activation and clearance.     

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.     

Involvement of adenosine A2A receptors in engulfment-dependent apoptotic cell suppression of inflammation

Efficient execution of apoptotic cell death followed by efficient clearance mediated by professional macrophages is a key mechanism in maintaining tissue homeostasis. Removal of apoptotic cells usually involves three central elements: 1) attraction of phagocytes via soluble "find me" signals, 2) recognition and phagocytosis via cell surface-presenting "eat me" signals, and 3) suppression or initiation of inflammatory responses depending on additional innate immune stimuli. Suppression of inflammation involves both direct inhibition of proinflammatory cytokine production and release of anti-inflammatory factors, which all contribute to the resolution of inflammation. In the current study, using wild-type and adenosine A(2A) receptor (A2AR) null mice, we investigated whether A2ARs, known to mediate anti-inflammatory signals in macrophages, participate in the apoptotic cell-mediated immunosuppression. We found that macrophages engulfing apoptotic cells release adenosine in sufficient amount to trigger A2ARs, and simultaneously increase the expression of A2ARs, as a result of possible activation of liver X receptor and peroxisome proliferators activated receptor δ. In macrophages engulfing apoptotic cells, stimulation of A2ARs suppresses the NO-dependent formation of neutrophil migration factors, such as macrophage inflammatory protein-2, using the adenylate cyclase/protein kinase A pathway. As a result, loss of A2ARs results in elevated chemoattractant secretion. This was evident as pronounced neutrophil migration upon exposure of macrophages to apoptotic cells in an in vivo peritonitis model. Altogether, our data indicate that adenosine is one of the soluble mediators released by macrophages that mediate engulfment-dependent apoptotic cell suppression of inflammation.     

Does nitric oxide play a role in maternal tolerance towards the foetus?

In pregnancy there occurs maternal tolerance to the foetus. Several mechanisms have been proposed to explain this phenomenon. The main immune population in the decidua are macrophages and natural killer cells, but with some "special" suppressor characteristics. There is also a predominant TH2 response. The non classical MCH type I HLA-G is expressed by trophoblasts and can suppress lymphomononuclear cytotoxicity. Other system to avoid the immune system is the expression of indoleamine-2,3-dioxygenase, that suppresses T cell activation by degrading tryptophan. Even though in the placenta there is a high production of nitric oxide, a well-known immune modulator, low attention has been paid to its role in maternal tolerance. There are many data showing that NO affects the IDO, CD95/CD95-L and the balance between TH1/TH2. Maybe NO could interact with several mechanisms at the same time, which could modify the tolerogenic activity depending on the concentration and the presence of other factors in the medium.     

Decidual macrophages and their roles at the maternal-fetal interface

The semi-allogeneic fetus, whose genome consists of maternally and paternally inherited alleles, must coexist with an active maternal immune system during its 9 months in utero. Macrophages are the second most abundant immune cell at the maternal-fetal interface, although populations and functions for these populations remain ill defined. We have previously reported two distinct subsets of CD14(+) decidual macrophages found to be present in first trimester decidual tissue, 20 percent CD11c(HI) and 68 percent CD11c(LO). Interestingly, CD11c(HI) decidual macrophages express genes associated with lipid metabolism, inflammation, and antigen presentation function and specifically upregulate CD1 molecules. Conversely, CD11c(LO) decidual macrophages express genes associated with extracellular matrix formation, muscle regulation, and tissue growth. The large abundance of CD11c(HI) decidual macrophages and their ability to process antigens more efficiently than CD11c(LO) macrophages suggests that CD11c(HI) macrophages may be important antigen processing and presenting cells at the maternal-fetal interface, while CD11c(LO) macrophages may perform necessary homeostatic functions during placental construction. Thus, macrophage heterogeneity may be an important and necessary division of labor that leads to both an induction of maternal immune cell tolerance to fetal antigens as well as basic homeostatic functions in human pregnancy.     

Role of surfactant proteins A,D, and C1q in the clearance of apoptotic cells in vivo and in vitro: calreticulin and CD91 as a common collectin receptor complex

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.     

Macrophages and dendritic cells use different Axl/Mertk/Tyro3 receptors in clearance of apoptotic cells

The clearance of apoptotic cells is important for regulating tissue homeostasis, inflammation, and autoimmune responses. The absence of receptor tyrosine kinases (Axl, Mertk, and Tyro3) results in widespread accumulation of apoptotic cells and autoantibody production in mice. In this report, we examine the function of the three family members in apoptotic cell clearance by different phagocytic cell types. Mertk elimination nearly abolished macrophage apoptotic cell phagocytosis; elimination of Axl, Tyro3, or both, reduced macrophage phagocytosis by approximately half, indicating that these also play a role. In contrast, apoptotic cell clearance in splenic and bone marrow-derived dendritic cells (DCs) is prolonged compared with macrophages and relied primarily on Axl and Tyro3. The slower ingestion may be due to lower DC expression of Axl and Tyro3 or absence of GAS6 expression, a known ligand for this receptor family. In vivo, phagocytosis of apoptotic material by retinal epithelial cells required Mertk. Unlike macrophages, there did not appear to be any role for Axl or Tyro3 in retinal homeostasis. Likewise, clearance of apoptotic thymocytes in vivo was dramatically reduced in mertk(kd) mice, but was normal in axl/tyro3(-/-) mice. Thus, cell and organ type specificity is clearly delineated, with DCs relying on Axl and Tyro3, retina and thymus requiring Mertk, and macrophages exhibiting an interaction that involves all three family members. Surprisingly, in macrophages, tyrosine phosphorylation of Mertk in response to apoptotic cells is markedly diminished from axl/tyro3(-/-) mice, suggesting that the interactions of these receptors by heterodimerization may be important in some cells.     

Glycodelin A, an immunomodulatory protein in the endometrium, inhibits proliferation and induces apoptosis in monocytic cells

Glycodelin A (GdA), is a lipocalin with an immunomodulatory role, secreted by the endometrium under progesterone regulation and proposed to play a role in protecting the fetus from maternal immune attack. Glycodelin A has an inhibitory effect on the proliferation of T cells and B cells and also on the activity of natural killer cells. We have earlier demonstrated that the inhibitory effect of glycodelin A on T cell proliferation is due to apoptosis induced in these cells through the caspase-dependent intrinsic mitochondrial pathway. Studies reported until now have shown that glycodelin modulates the adaptive immune responses. We, therefore, decided to look at its effect, if any, on the innate immune system. The effect of glycodelin on monocytes was studied using human monocytic cell lines, THP1 and U937, and primary human monocytes as model systems. We demonstrated that glycodelin inhibited the proliferation of THP1 and U937 and induced apoptosis in these cells as well as in primary monocytes. We found that this signaling was caspase-independent but followed the intrinsic mitochondrial pathway of apoptosis. No effect of glycodelin was seen on the phagocytic ability of monocytes post-differentiation into macrophages. These observations suggest that, at the fetomaternal interface, glycodelin plays a protective role by deleting the monocytes that could become pro-inflammatory. Importantly, leaving the macrophages untouched to carry on with efficient clearance of the apoptotic cells.     

Selective Depletion of Eosinophils or Neutrophils in Mice Impacts the Efficiency of Apoptotic Cell Clearance in the Thymus

Developing thymocytes undergo a rigorous selection process to ensure that the mature T cell population expresses a T cell receptor (TCR) repertoire that can functionally interact with major histocompatibility complexes (MHC). Over 90% of thymocytes fail this selection process and die. A small number of macrophages within the thymus are responsible for clearing the large number of dying thymocytes that must be continuously cleared. We studied the capacity of thymic macrophages to clear apoptotic cells under acute circumstances. This was done by synchronously inducing cell death in the thymus and then monitoring the clearance of apoptotic thymocytes. Interestingly, acute cell death was shown to recruit large numbers of CD11b⁺ cells into the thymus. In the absence of a minor CSF-1 dependent population of macrophages, the recruitment of these CD11b⁺ cells into the thymus was greatly reduced and the clearance of apoptotic cells was disrupted. To assess a possible role for the CD11b⁺ cells in the clearance of apoptotic cells, we analyzed mice deficient for eosinophils and mice with defective trafficking of neutrophils. Failure to attract either eosinophils or neutrophils to the thymus resulted in the impaired clearance of apoptotic cells. These results suggested that there is crosstalk between cells of the innate immune system that is necessary for maximizing the efficiency of apoptotic cell removal.     

Engulfment and clearance of apoptotic cells based on a GlcNAc-binding lectin-like property of surface vimentin

The clearance of apoptotic cells is important to maintain tissue homeostasis. The engulfment of apoptotic cells is performed by professional phagocytes, such as macrophages, and also by non-professional phagocytes, such as mesenchymal cells. Here, we show that vimentin, a cytoskeletal protein, functions as an engulfment receptor on neighboring phagocytes, which recognize O-linked β-N-acetylglucosamine (O-GlcNAc)-modified proteins from apoptotic cells as "eat me" ligands. Previously, we reported that vimentin possesses a GlcNAc-binding lectin-like property on cell surface. However, the physiological relevance of the surface localization and GlcNAc-binding property of vimentin remained unclear. In the present study, we observed that O-GlcNAc proteins from apoptotic cells interacted with the surface vimentin of neighboring phagocytes and that this interaction induced serine 71-phosphorylation and recruitment of vimentin to the cell surface of the neighboring phagocytes. Moreover, tetrameric vimentin that was disassembled by serine 71-phosphorylation possessed a GlcNAc-binding activity and was localized to the cell surface. We demonstrated our findings in vimentin-expressing common cell lines such as HeLa cells. Furthermore, during normal developmental processes, the phagocytic engulfment and clearance of apoptotic footplate cells in mouse embryos was mediated by the interaction of surface vimentin with O-GlcNAc proteins. Our results suggest a common mechanism for the clearance of apoptotic cells, through the interaction of surface vimentin with O-GlcNAc-modified proteins.     

A Role for Potassium Permeability in the Recognition,Clearance, and Anti-inflammatory Effects of Apoptotic Cells

The benefits of programmed cell death by apoptosis are the safe and efficient clearance of damaged, infected, or surplus cells, primarily mediated by tissue-resident macrophages or tissue-infiltrating blood monocytes that differentiate into macrophages. Microglial cells are macrophages of the brain parenchyma, important immune surveillance cells that respond to various injuries and diseases of the brain. It is often stated that how a macrophage interacts with an apoptotic cell defines subsequent inflammatory responses, i.e., will engulfment be beneficial or detrimental for tissue repair, regeneration, and immunity. Our focus has been to better understand how macrophages discriminate between living and dying cells. Following our initial findings with platelet endothelial cell adhesion molecule (PECAM)-1, our studies have revealed a key role for potassium ion permeability in regulating integrin-dependent binding of apoptotic cells by macrophages and their subsequent response to proinflammatory stimuli. Specifically, apoptotic cells represent a depolarizing stimulus for macrophages where PECAM-1-mediated cell–cell interactions delay subsequent membrane repolarization. It is salient that potassium leak represents an early feature of cells destined to die by apoptosis that could trigger depolarization of macrophages that lie in close apposition. We speculate that how a tissue-resident macrophage responds to strong depolarizing stimuli has wider implications for inflammation and autoimmunity.     

Identification of TLT2 as an engulfment receptor for apoptotic cells

Clearance of apoptotic cells (efferocytosis) is critical to the homeostasis of the immune system by restraining inflammation and autoimmune response to intracellular Ags released from dying cells. TLRs-mediated innate immunity plays an important role in pathogen clearance and in regulation of the adaptive immune response. However, the regulation of efferocytosis by activation of TLRs has not been well characterized. In this study, we found that activation of TLR3 or TLR9, but not of TLR2, enhances engulfment of apoptotic cells by macrophages. We found that the activation of TLR3 upregulates the expression of triggering receptor expressed on myeloid cells (TREM)-like protein 2 (TLT2), a member of the TREM receptor family, on the surface of macrophages. Blocking TLT2 on the macrophage surface by either specific anti-TLT2 Ab or soluble TLT2 extracellular domain attenuated the enhanced ability of macrophages with TLR3 activation to engulf apoptotic cells. To the contrary, overexpression of TLT2 increased the phagocytosis of apoptotic cells. We found that TLT2 specifically binds to phosphatidylserine, a major "eat me" signal that is exposed on the surface of apoptotic cells. Furthermore, we found that TLT2 mediates phagocytosis of apoptotic cells in vivo. Thus, our studies identified TLT2 as an engulfment receptor for apoptotic cells. Our data also suggest a novel mechanism by which TREM receptors regulate inflammation and autoimmune response.     

Apoptotic neutrophils containing Staphylococcus epidermidis stimulate macrophages to release the proinflammatory cytokines tumor necrosis factor-alpha and interleukin-6.

Staphylococcus epidermidis infections are usually nosocomial and involve colonization of biomaterials. The immune defense system cannot efficiently control the bacteria during these infections, which often results in protracted chronic inflammation, in which a key event is disturbed removal of neutrophils by tissue macrophages. While ingesting uninfected apoptotic neutrophils, macrophages release anti-inflammatory cytokines that lead to resolution of inflammation. In clinical studies, we have previously found elevated levels of the proinflammatory cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 in synovial fluid from prostheses infected with coagulase negative staphylococci. We show that macrophages phagocytosing apoptotic neutrophils containing S. epidermidis released TNF-alpha and interleukin-6, whereas macrophages phagocytosing spontaneously apoptotic neutrophils did not. This difference was not due to dissimilar phagocytic capacities, because macrophages ingested both types of neutrophils to the same extent. The activation was induced mainly by the apoptotic neutrophils themselves, not by the few remaining extracellular bacteria. Macrophages were not activated by apoptotic neutrophils that contained paraformaldehyde-killed S. epidermidis. Proinflammatory reactions induced by clearance of apoptotic neutrophils containing S. epidermidis might represent an important mechanism to combat the infective agent. This activation of macrophages may contribute to the development of chronic inflammation instead of inflammation resolution.