Apoptotic cells are cleared by phagocytosis during development, homeostasis, and pathology. However, it is still unclear how necrotic cells are removed. We compared the phagocytic uptake by macrophages of variants of L929sA murine fibrosarcoma cells induced to die by tumor necrosis factor-induced necrosis or by Fas-mediated apoptosis. We show that apoptotic and necrotic cells are recognized and phagocytosed by macrophages, whereas living cells are not. In both cases, phagocytosis occurred through a phosphatidylserine-dependent mechanism, suggesting that externalization of phosphatidylserine is a general trigger for clearance by macrophages. However, uptake of apoptotic cells was more efficient both quantitatively and kinetically than phagocytosis of necrotic cells. Electron microscopy showed clear morphological differences in the mechanisms used by macrophages to engulf necrotic and apoptotic cells. Apoptotic cells were taken up as condensed membrane-bound particles of various sizes rather than as whole cells, whereas necrotic cells were internalized only as small cellular particles after loss of membrane integrity. Uptake of neither apoptotic nor necrotic L929 cells by macrophages modulated the expression of proinflammatory cytokines by the phagocytes.
Various types of phagocytes mediate the clearance of apoptotic cells. We previously reported that human and murine high endothelial venule (HEV) cells ingest apoptotic cells. In this report, we examined endothelial cell fucoidin receptor-mediated phagocytosis using a murine endothelial cell model mHEV. mHEV cell recognition of apoptotic leukocytes was blocked by fucoidin but not by other phagocytic receptor inhibitors such as mannose, fucose, N-acetylglucosamine, phosphatidylserine (PS), or blocking anti-PS receptor antibodies. Thus, the mHEV cells used fucoidin receptors for recognition and phagocytosis of apoptotic leukocytes. The fucoidin receptor-mediated endothelial cell phagocytosis was specific for apoptotic leukocytes, as necrotic cells were not ingested. This is in contrast to macrophages, which ingest apoptotic and necrotic cells. Endothelial cell phagocytosis of apoptotic cells did not alter viable lymphocyte migration across these endothelial cells. Antibody blocking of CD44 and α4 integrin on the apoptotic leukocyte inhibited this endothelial cell phagocytosis, suggesting a novel function for these adhesion molecules in the removal of apoptotic targets. The removal of apoptotic leukocytes by endothelial cells may protect the microvasculature, thus ensuring that viable lymphocytes can successfully migrate into tissues.
lectin; carbohydrate; cell adhesion molecules; phosphatidylserine; endothelial cells
Effective clearance of apoptotic cells by macrophages is essential for immune homeostasis. The transcriptional pathways that allow macrophages to sense and respond to apoptotic cells are poorly defined. We demonstrate here that LXR signaling is important for both apoptotic cell clearance and the maintenance of immune tolerance. Apoptotic cell engulfment activates LXR and thereby induces the expression of Mer, a receptor tyrosine kinase critical for phagocytosis. LXR null macrophages exhibit a selective defect in phagocytosis of apoptotic cells and an aberrant pro-inflammatory response to them. As a consequence of these defects, mice lacking LXRs manifest a breakdown in self-tolerance and develop autoantibodies and autoimmune glomerulonephritis. Treatment with an LXR agonist ameliorates disease progression in mouse models of Lupus-like autoimmunity. Thus, activation of LXR by apoptotic cells engages a virtuous cycle that promotes their own clearance and couples engulfment to the suppression of inflammatory pathways.
Interaction of macrophages with apoptotic cells involves multiple steps including recognition, tethering, phagocytosis, and anti-inflammatory macrophage responses. Defective apoptotic cell clearance is associated with pathogenesis of autoimmune disease. CD14 is a surface receptor that functions in vitro in the removal of apoptotic cells by human and murine macrophages, but its mechanism of action has not been defined. Here, we demonstrate that CD14 functions as a macrophage tethering receptor for apoptotic cells. Significantly, CD14−/− macrophages in vivo are defective in clearing apoptotic cells in multiple tissues, suggesting a broad role for CD14 in the clearance process. However, the resultant persistence of apoptotic cells does not lead to inflammation or increased autoantibody production, most likely because, as we show, CD14−/− macrophages retain the ability to generate anti-inflammatory signals in response to apoptotic cells. We conclude that CD14 plays a broad tethering role in apoptotic cell clearance in vivo and that apoptotic cells can persist in the absence of proinflammatory consequences.
The distinction between physiological (apoptotic) and pathological
(necrotic) cell deaths reflects mechanistic differences in cellular
disintegration and is of functional significance with respect to the
outcomes that are triggered by the cell corpses. Mechanistically,
apoptotic cells die via an active and ordered pathway; necrotic deaths,
conversely, are chaotic and passive. Macrophages and other phagocytic
cells recognize and engulf these dead cells. This clearance is believed
to reveal an innate immunity, associated with inflammation in cases of
pathological but not physiological cell deaths. Using objective and
quantitative measures to assess these processes, we find that
macrophages bind and engulf native apoptotic and necrotic cells to
similar extents and with similar kinetics. However, recognition of
these two classes of dying cells occurs via distinct and noncompeting
mechanisms. Phosphatidylserine, which is externalized on both apoptotic
and necrotic cells, is not a specific ligand for the recognition of
either one. The distinct modes of recognition for these different
corpses are linked to opposing responses from engulfing macrophages.
Necrotic cells, when recognized, enhance proinflammatory responses of
activated macrophages, although they are not sufficient to trigger
macrophage activation. In marked contrast, apoptotic cells profoundly
inhibit phlogistic macrophage responses; this represents a
cell-associated, dominant-acting anti-inflammatory signaling activity
acquired posttranslationally during the process of physiological cell
The mammalian ATP-binding cassette transporters A1 and A7 (ABCA1 and -A7) show sequence similarity to CED-7, a Caenorhabditis elegans gene that mediates the clearance of apoptotic cells. Using RNA interference or gene targeting, we show that knock down of macrophage ABCA7 but not -A1 results in defective engulfment of apoptotic cells. In response to apoptotic cells, ABCA7 moves to the macrophage cell surface and colocalizes with the low-density lipoprotein receptor–related protein 1 (LRP1) in phagocytic cups. The cell surface localization of ABCA7 and LRP1 is defective in ABCA7-deficient cells. C1q is an opsonin of apoptotic cells that acts via phagocyte LRP1 to induce extracellular signal–regulated kinase (ERK) signaling. We show that ERK signaling is required for phagocytosis of apoptotic cells and that ERK phosphorylation in response to apoptotic cells or C1q is defective in ABCA7-deficient cells. These studies reveal a major role of ABCA7 and not -A1 in the clearance of apoptotic cells and therefore suggest that ABCA7 is an authentic orthologue of CED-7.
Atherosclerotic plaques that are prone to disruption and acute thrombotic vascular events are characterized by large necrotic cores. Necrotic cores result from the combination of macrophage apoptosis and defective phagocytic clearance (efferocytosis) of these apoptotic cells. We previously showed that macrophages with tyrosine kinase-defective Mertk receptor (MertkKD) have a defect in phagocytic clearance of apoptotic macrophages in vitro. Herein we test the hypothesis that the MertkKD mutation would result in increased accumulation of apoptotic cells and promote necrotic core expansion in a mouse model of advanced atherosclerosis.
Methods and Results
MertkKD;Apoe−/− mice and control Apoe−/− mice were fed a Western-type diet for 10 or 16 wks, and aortic root lesions were analyzed for apoptosis and plaque necrosis. We found that the plaques of the MertkKD;Apoe−/− mice had a significant increase in TUNEL-positive apoptotic cells. Most importantly, there were more non-macrophage-associated apoptotic cells in the MertkKD lesions, consistent with defective efferocytosis. The more advanced (16-wk) MertkKD;Apoe−/− plaques were more necrotic, consistent with a progression from apoptotic cell accumulation to plaque necrosis in the setting of a defective efferocytosis receptor.
In a mouse model of advanced atherosclerosis, mutation of the phagocytic Mertk receptor promotes the accumulation of apoptotic cells and the formation of necrotic plaques. These data are consistent with the notion that a defect in an efferocytosis receptor can accelerate the progression of atherosclerosis and suggest a novel therapeutic target to prevent advanced plaque progression and its clinical consequences.
Atherosclerosis-Pathophysiology; Apoptosis; Phagocytosis; Animal models of human disease
Cathelicidins are CHDP with essential roles in innate host defense but also more recently associated with the pathogenesis of certain chronic diseases. These peptides have microbicidal potential and the capacity to modulate innate immunity and inflammatory processes. PMN are key innate immune effector cells with pivotal roles in defense against infection. The appropriate regulation of PMN function, death, and clearance is critical to innate immunity, and dysregulation is implicated in disease pathogenesis. The efferocytosis of apoptotic PMN, in contrast to necrotic cells, is proposed to promote the resolution of inflammation. We demonstrate that the human cathelicidin LL-37 induced rapid secondary necrosis of apoptotic human PMN and identify an essential minimal region of LL-37 required for this activity. Using these LL-37-induced secondary necrotic PMN, we characterize the consequence for macrophage inflammatory responses. LL-37-induced secondary necrosis did not inhibit PMN ingestion by monocyte-derived macrophages and in contrast to expectation, was not proinflammatory. Furthermore, the anti-inflammatory effects of apoptotic PMN on activated macrophages were retained and even potentiated after LL-37-induced secondary necrosis. However, this process of secondary necrosis did induce the release of potentially harmful PMN granule contents. Thus, we suggest that LL-37 can be a potent inducer of PMN secondary necrosis during inflammation without promoting macrophage inflammation but may mediate host damage through PMN granule content release under chronic or dysregulated conditions.
cationic host defense peptide; antimicrobial peptide; innate immunity; inflammation; efferocytosis
Defective clearance of apoptotic cells has been shown in systemic lupus erythematosus (SLE) and is postulated to enhance autoimmune responses by increasing access to intracellular autoantigens. Until now, research has emphasized inherited rather than acquired impairment of apoptotic cell engulfment in the pathogenesis of SLE. Here, we confirm previous results that efficient removal of apoptotic cells (efferocytosis) is bolstered in the presence of wild type mouse serum, through the C3 deposition on the apoptotic cell surface. In contrast, sera from three mouse models of SLE, MerKD, MRLlpr and NZBWF1, did not support and in fact actively inhibited apoptotic cell uptake. IgG autoantibodies were responsible for the inhibition, through the blockade of C3 recognition by macrophages. Consistent with this, IgG removal reversed the inhibitory activity within autoimmune serum and purified autoimmune IgG blocked both the detection of C3 on apoptotic cells and C3-dependent efferocytosis. Sera from SLE patients demonstrated elevated anti-C3b IgG that blocked detection of C3 on apoptotic cells, activity that was not found in healthy controls or patients with rheumatoid arthritis, nor in mice prior to the onset of autoimmunity. We propose that the suppression of apoptotic cell disposal by antibodies against deposited C3 may contribute to increasing severity and/or exacerbations in SLE.
Phagocytic removal of apoptotic cells occurs efficiently in vivo such that even in tissues with significant apoptosis, very few apoptotic cells are detectable1. This is thought to be due to the release of find-me signals by apoptotic cells that recruit motile phagocytes such as monocytes, macrophages, and dendritic cells, leading to the prompt clearance of the dying cells2. However, the identity and in vivo relevance of such find-me signals are not well understood. Here, through several lines of evidence, we identify extracellular nucleotides as a critical apoptotic cell find-me signal. We demonstrate the caspase-dependent release of ATP and UTP (in equimolar quantities) during the early stages of apoptosis by primary thymocytes and cell lines. Purified nucleotides at these concentrations were sufficient to induce monocyte recruitment comparable to apoptotic cell supernatants. Enzymatic removal of ATP and UTP (by apyrase or ectopic CD39 expression) abrogated the ability of apoptotic cell supernatants to recruit monocytes in vitro and in vivo. We then identified the ATP/UTP receptor P2Y2 as a critical sensor of nucleotides released by apoptotic cells using RNAi depletion studies in monocytes, and macrophages from P2Y2-null mice3. The in vivo relevance of nucleotides in apoptotic cell clearance was revealed by two approaches. First, in a murine air-pouch model, apoptotic cell supernatants induced a three-fold greater recruitment of monocytes and macrophages compared to supernatants from healthy cells; this recruitment was abolished by depletion of nucleotides and significantly decreased in P2Y2−/− mice. Second, clearance of apoptotic thymocytes was significantly impaired by either depletion of nucleotides or interference with P2Y receptor function (by pharmacological inhibition, or in P2Y2−/− mice). These results identify nucleotides as a critical find-me cue released by apoptotic cells to promote P2Y2-dependent phagocyte recruitment, and provide strong evidence for a clear relationship between a find-me signal and efficient corpse clearance in vivo.
Exposure of phosphatidylserine (PS) on the cell surface occurs early during apoptosis and serves as a recognition signal for phagocytes. Clearance of apoptotic cells by a membrane PS receptor is one of the critical anti-inflammatory functions of macrophages. However, the PS binding receptors and their recognition mechanisms have not been fully investigated. Recently, we reported that stabilin-2 is a PS receptor that mediates the clearance of apoptotic cells, thus releasing the anti-inflammatory cytokine, transforming growth factor β. In this study, we showed that epidermal growth factor (EGF)-like domain repeats (EGFrp) in stabilin-2 can directly and specifically recognize PS. The EGFrps also competitively impaired apoptotic cell uptake by macrophages in in vivo models. We also showed that calcium ions are required for stabilin-2 to mediate phagocytosis via EGFrp. Interestingly, at least four tandem repeats of EGF-like domains were required to recognize PS, and the second atypical EGF-like domain in EGFrp was critical for calcium-dependent PS recognition. Considering that PS itself is an important target molecule for both apoptotic cells and nonapoptotic cells during various cellular processes, our results should help elucidate the molecular mechanism by which apoptotic cell clearance in the human body occurs and also have implications for targeting PS externalization of nonapoptotic cells.
The complications of atherosclerosis are a major cause of death and disability in Type 2 diabetes. Defective clearance of apoptotic cells by macrophages (efferocytosis) is thought to lead to increased necrotic core formation and inflammation in atherosclerotic lesions.
To determine if there is defective efferocytosis in a mouse model of obesity and atherosclerosis.
Methods and Results
We quantified efferocytosis in peritoneal macrophages and in atherosclerotic lesions of obese ob/ob or ob/ob;Ldlr−/− mice and littermate controls. Peritoneal macrophages from ob/ob and ob/ob;Ldlr−/− mice showed impaired efferocytosis, reflecting defective PI3K activation during uptake of apoptotic cells. Membrane lipid composition of ob/ob and ob/ob;Ldlr−/− macrophages showed an increased content of saturated fatty acids (FAs), and decreased n-3 FAs ((Eicosapentaenoic acid (EPA) and Docosahexaenoic acid (DHA)) compared to controls. A similar defect in efferocytosis was induced by treating control macrophages with saturated free FA (FFA)/BSA complexes, while the defect in ob/ob macrophages was reversed by treatment with EPA/BSA or by feeding ob/ob mice a fish oil diet rich in n-3 FAs. There was also defective macrophage efferocytosis in atherosclerotic lesions of ob/ob;Ldlr−/− mice and this was reversed by a fish oil-rich diet.
The findings suggest that in obesity and Type 2 diabetes elevated levels of saturated FAs and/or decreased levels of n-3 FAs contribute to decreased macrophage efferocytosis. Beneficial effects of fish oil diets in atherosclerotic cardiovascular disease may involve improvements in macrophage function related to reversal of defective efferocytosis, and could be particularly important in Type 2 diabetes and obesity.
Efferocytosis; Macrophages; Atherosclerosis; Fatty Acids; Mouse
c-Jun activation by mitogen-activated protein kinases has been implicated in various cellular signal responses. We investigated how JNK and c-Jun contribute to neuronal differentiation, cell survival, and apoptosis. In differentiated PC12 cells, JNK signaling can induce apoptosis and c-Jun mediates this response. In contrast, we show that in PC12 cells that are not yet differentiated, the AP-1 family member ATF-2 and not c-Jun acts as an executor of apoptosis. In this context c-Jun expression protects against apoptosis and triggers neurite formation. Thus, c-Jun has opposite functions before and after neuronal differentiation. These findings suggest a model in which the balance between ATF-2 and Jun activity in PC12 cells governs the choice between differentiation towards a neuronal fate and an apoptotic program. Further analysis of c-Jun mutants showed that the differentiation response requires functional dimerization and DNA-binding domains and that it is stimulated by phosphorylation in the transactivation domain. In contrast, c-Jun mutants incompetent for DNA binding or dimerization and also mutants lacking JNK binding and phosphorylation sites that cannot elicit neuronal differentiation efficiently protect PC12 cells from apoptosis. Hence, the protective role of c-Jun appears to be mediated by an unconventional mechanism that is separable from its function as a classical AP-1 transcription factor.
Atherothrombotic vascular disease is the major cause of death and disability in obese and diabetic subjects with insulin resistance. Although increased systemic risk factors in the setting of insulin resistance contribute to this problem, it is likely exacerbated by direct effects of insulin resistance on the arterial wall cells that participate in atherosclerosis. A critical process in the progression of atherosclerotic lesions to those that cause clinical disease is necrotic breakdown of plaques. Plaque necrosis, which is particularly prominent in the lesions of diabetics, is caused by the combination of macrophage apoptosis and defective clearance, or efferocytosis, of the apoptotic macrophages. One cause of macrophage apoptosis in advanced plaques is activation of a pro-apoptotic branch of the endoplasmic reticulum stress pathway known as the Unfolded Protein Response (UPR). Macrophages have a functional insulin receptor signal transduction pathway, and down regulation of this pathway in the setting insulin resistance enhances UPR-induced apoptosis. Moreover, other aspects of the obesity/insulin-resistance syndrome may adversely affect efferocytosis. These processes may therefore provide an important mechanistic link among insulin resistance, plaque necrosis, and atherothrombotic vascular disease and suggest novel therapeutic approaches to this expanding health problem.
atherosclerosis; insulin resistance; diabetes; macrophages; apoptosis; efferocytosis; plaque necrosis; ER stress; Unfolded Protein Response
Obesity and type 2 diabetes are associated with chronic inflammation. Adiponectin is an adipocyte-derived hormone with antidiabetic and antiinflammatory actions. Here, we demonstrate what we believe to be a previously undocumented activity of adiponectin, facilitating the uptake of early apoptotic cells by macrophages, an essential feature of immune system function. Adiponectin-deficient (APN-KO) mice were impaired in their ability to clear apoptotic thymocytes in response to dexamethasone treatment, and these animals displayed a reduced ability to clear early apoptotic cells that were injected into their intraperitoneal cavities. Conversely, adiponectin administration promoted the clearance of apoptotic cells by macrophages in both APN-KO and wild-type mice. Adiponectin overexpression also promoted apoptotic cell clearance and reduced features of autoimmunity in lpr mice whereas adiponectin deficiency in lpr mice led to a further reduction in apoptotic cell clearance, which was accompanied by exacerbated systemic inflammation. Adiponectin was capable of opsonizing apoptotic cells, and phagocytosis of cell corpses was mediated by the binding of adiponectin to calreticulin on the macrophage cell surface. We propose that adiponectin protects the organism from systemic inflammation by promoting the clearance of early apoptotic cells by macrophages through a receptor-dependent pathway involving calreticulin.
Ingestion of apoptotic cells in vitro by macrophages induces TGF-β1 secretion, resulting in an anti-inflammatory effect and suppression of proinflammatory mediators. Here, we show in vivo that direct instillation of apoptotic cells enhanced the resolution of acute inflammation. This enhancement appeared to require phosphatidylserine (PS) on the apoptotic cells and local induction of TGF-β1. Working with thioglycollate-stimulated peritonea or LPS-stimulated lungs, we examined the effect of apoptotic cell uptake on TGF-β1 induction. Viable or opsonized apoptotic human Jurkat T cells, or apoptotic PLB-985 cells, human monomyelocytes that do not express PS during apoptosis, failed to induce TGF-β1. PS liposomes, or PS directly transferred onto the PLB-985 surface membranes, restored the TGF-β1 induction. Apoptotic cell instillation into LPS-stimulated lungs reduced proinflammatory chemokine levels in the bronchoalveolar lavage fluid (BALF). Additionally, total inflammatory cell counts in the BALF were markedly reduced 1–5 days after apoptotic cell instillation, an effect that could be reversed by opsonization or coinstillation of TGF-β1 neutralizing antibody. This reduction resulted from early decrease in neutrophils and later decreases in lymphocytes and macrophages. In conclusion, apoptotic cell recognition and clearance, via exposure of PS and ligation of its receptor, induce TGF-β1 secretion, resulting in accelerated resolution of inflammation.
C-reactive protein (CRP) is a serum protein that is massively induced as part of the innate immune response to infection and tissue injury. As CRP has been detected in damaged tissues and is known to activate complement, we assessed whether apoptotic lymphocytes bound CRP and determined the effect of binding on innate immunity. CRP bound to apoptotic cells in a Ca2+-dependent manner and augmented the classical pathway of complement activation but protected the cells from assembly of the terminal complement components. Furthermore, CRP enhanced opsonization and phagocytosis of apoptotic cells by macrophages associated with the expression of the antiinflammatory cytokine transforming growth factor β. The antiinflammatory effects of CRP required C1q and factor H and were not effective once cells had become necrotic. These observations demonstrate that CRP and the classical complement components act in concert to promote noninflammatory clearance of apoptotic cells and may help to explain how deficiencies of the classical pathway and certain pentraxins lead to impaired handling of apoptotic cells and increased necrosis with the likelihood of immune response to self.
apoptosis; C-reactive protein; complement; macrophages; autoimmunity
Macrophages rapidly engulf apoptotic cells to limit the release of noxious cellular contents and to restrict autoimmune responses against self antigens. Although factors participating in recognition and engulfment of apoptotic cells have been identified, the transcriptional basis for the sensing and silently disposing of apoptotic cells is unknown. Here we show that peroxisome proliferator activated receptor-δ (PPAR-δ) is induced when macrophages engulf apoptotic cells and functions as a transcriptional sensor of dying cells. Genetic deletion of PPAR-δ decreases expression of opsonins, such as C1qb, resulting in impairment of apoptotic cell clearance and reduction in anti-inflammatory cytokine production. This increases autoantibody production and predisposes global and macrophage-specific PPARd−/− mice to autoimmune kidney disease, a phenotype resembling the human disease systemic lupus erythematosus. Thus, PPAR-δ plays a pivotal role in orchestrating the timely disposal of apoptotic cells by macrophages, ensuring that tolerance to self is maintained.
Survival and death of lymphocytes is regulated by the balance between pro- and anti-apoptotic members of the Bcl-2 family, and this is coordinated with the control of cell cycling and differentiation. Bim, a pro-apoptotic BH3-only member of the Bcl-2 family, can be regulated by MEK/ERK-mediated phosphorylation, which affects its binding to pro-survival Bcl-2 family members and its turnover. We investigated Bim modifications in mouse B and T lymphoid cells after exposure to apoptotic stimuli and during mitogenic activation. Treatment with ionomycin or cytokine withdrawal caused an elevation in BimEL, the most abundant Bim isoform. In contrast, in mitogenically stimulated T and B cells, BimEL was rapidly phosphorylated and its levels declined. Pharmacological inhibitors of MEK/ERK-signaling prevented both of these changes in Bim, reduced proliferation and triggered apoptosis of mitogen-stimulated T and B cells. Remarkably, loss of Bim prevented this cell killing but did not restore cell cycling. These results show that during mitogenic stimulation of T and B lymphocytes MEK/ERK signaling is critical for two distinct processes, cell survival, mediated (at least in part) through phosphorylation and consequent inhibition of Bim, and cell cycling, which proceeds independently of Bim inactivation.
T cells; B cells; cell activation; apoptosis; signal transduction
Phagocytic clearance of apoptotic cells by macrophages is an essential part in the resolution of inflammation. It coincides with activation of repair mechanisms, including accumulation of extracellular matrix. A possible link between clearance of apoptotic debris and accumulation of extracellular matrix has not been investigated. Production of collagen was measured in primary fibroblasts co-cultured with macrophages. Ingestion of apoptotic cells by monocyte-derived macrophages led to upregulation of collagen. Direct contact between macrophages and fibroblasts was not required for collagen upregulation. Macrophages produced TGF- β following ingestion of apoptotic cells, but the levels of this cytokine were lower than those required for a significant upregulation of collagen. Simultaneously, the levels of TGF-β-induced (TGFBI), or keratoepithelin/BIGH3, mRNA and protein were increased. In contrast, primary alveolar macrophages stimulated collagen production without exposure to apoptotic cells; there was no further increase in the levels of TGFBI, mRNA or protein, or collagen after ingestion of apoptotic cells. Stimulation of fibroblasts with TGFBI downregulated MMP14 levels, decreased DNA binding by p53, increased DNA binding by PU.1, and upregulated collagen protein but not mRNA levels. Overexpression of MMP14 or p53, or siRNA-mediated inhibition of PU.1 led to an increase in MMP14 and a decline in collagen levels, whereas siRNA-mediated inhibition of MMP14 led to elevation of collagen levels. In conclusion, monocyte-derived but not alveolar macrophages produce TGFBI following ingestion of apoptotic cells, leading to downregulation of MMP14 levels in fibroblasts through a mechanism involving p53 and PU.1, and to subsequent accumulation of collagen.
cytokines; fibroblasts; matrix metalloproteinases; macrophages; apoptosis; collagen; fibrosis
A high intracellular bacillary load of Mycobacterium tuberculosis in macrophages induces an atypical lysosomal cell death with early features of apoptosis that progress to necrosis within hours. Unlike classical apoptosis, this cell death mode does not appear to diminish M. tuberculosis viability. We previously reported that culturing heavily infected macrophages with naïve macrophages produced an antimicrobial effect, but only if naïve macrophages were added during the pre-necrotic phase of M. tuberculosis-induced cell death. In the present study we investigated the mechanism of antimicrobial activity in co-cultures, anticipating that efferocytosis of bacilli in apoptotic bodies would be required. Confocal microscopy revealed frustrated phagocytosis of M. tuberculosis-infected macrophages with no evidence that significant numbers of bacilli were transferred to the naïve macrophages. The antimicrobial effect of naïve macrophages was retained when they were separated from infected macrophages in transwells, and conditioned co-culture supernatants transferred antimicrobial activity to cultures of infected macrophages alone. Antimicrobial activity in macrophage co-cultures was abrogated when the naïve population was deficient in IL-1 receptor or when the infected population was deficient in inducible nitric oxide synthase. The participation of nitric oxide suggested a conventional antimicrobial mechanism requiring delivery of bacilli to a late endosomal compartment. Using macrophages expressing GFP-LC3 we observed the induction of autophagy specifically by a high intracellular load of M. tuberculosis. Bacilli were identified in LC3-positive compartments and LC3-positive compartments were confirmed to be acidified and LAMP1 positive. Thus, the antimicrobial effect of naïve macrophages acting on M. tuberculosis in heavily-infected macrophages is contact-independent. Interleukin-1 provides an afferent signal that induces an as yet unidentified small molecule which promotes nitric oxide-dependent antimicrobial activity against bacilli in autolysosomes of heavily infected macrophages. This cooperative, innate antimicrobial interaction may limit the maximal growth rate of M. tuberculosis prior to the expression of adaptive immunity in pulmonary tuberculosis.
In cardiovascular disorders including advanced atherosclerosis and myocardial infarction (MI), increased cell death and tissue destabilization is associated with recruitment of inflammatory monocyte subsets that give rise to differentiated macrophages. These phagocytic cells clear necrotic and apoptotic bodies and promote inflammation resolution and tissue remodeling. The capacity of macrophages for phagocytosis of apoptotic cells (efferocytosis), clearance of necrotic cell debris, and repair of damaged tissue are challenged and modulated by local cell stressors that include increased protease activity, oxidative stress, and hypoxia. The effectiveness, or lack thereof, of phagocyte-mediated clearance, in turn is linked to active inflammation resolution signaling pathways, susceptibility to atherothrombosis and potentially, adverse post MI cardiac remodeling leading to heart failure. Previous reports indicate that in advanced atherosclerosis, defective efferocytosis is associated with atherosclerotic plaque destabilization. Post MI, the role of phagocytes and clearance in the heart is less appreciated. Herein we contrast the roles of efferocytosis in atherosclerosis and post MI and focus on how targeted modulation of clearance and accompanying resolution and reparative signaling may be a strategy to prevent heart failure post MI.
macrophage; phagocytosis; cardiovascular; myocardial infarction; clearance; hypoxia
BACKGROUND: There are two fundamental forms of cell death: apoptosis and necrosis. Molecular studies of cell death thus far favor a model in which apoptosis and necrosis share very few molecular regulators. It appears that apoptotic processes triggered by a variety of stimuli converge on the activation of a member of the caspase family, such as caspase 3, which leads to the execution of apoptosis. It has been suggested that blocking of caspase activation in an apoptotic process may divert cell death to a necrotic demise, suggesting that apoptosis and necrosis may share some upstream events. Activation of caspase is preceded by the release of mitochondrial cytochrome C. MATERIALS AND METHODS: We first studied cell death induced by beta-lapachone by MTT and colony-formation assay. To determine whether the cell death induced by beta-lapachone occurs through necrosis or apoptosis, we used the PI staining procedure to determine the sub-G1 fraction and the Annexin-V staining for externalization of phophatidylserine. We next compared the release of mitochondrial cytochrome C in apoptosis and necrosis. Mitochondrial cytochrome C was determined by Western blot analysis. To investigate changes in mitochondria that resulted in cytochrome C release, the mitochondrial membrane potential (delta psi) was analyzed by the accumulation of rhodamine 123, a membrane-permeant cationic fluorescent dye. The activation of caspase in apoptosis and necrosis were measured by using a profluorescent substrate for caspase-like proteases, PhiPhiLuxG6D2. RESULTS: beta-lapachone induced cell death in a spectrum of human carcinoma cells, including nonproliferating cells. It induced apoptosis in human ovary, colon, and lung cancer cells, and necrotic cell death in four human breast cancer cell lines. Mitochondrial cytochrome C release was found in both apoptosis and necrosis. This cytochrome C release occurred shortly after beta-lapachone treatment when cells were fully viable by trypan blue exclusion and MTT assay, suggesting that cytochrome C release is an early event in beta-lapachone induced apoptosis as well as necrosis. The mitochondrial cytochrome C release induced by beta-lapachone is associated with a decrease in mitochondrial transmembrane potential (delta psi). There was activation of caspase 3 in apoptotic cell death, but not in necrotic cell death. This lack of activation of CPP 32 in human breast cancer cells is consistent with the necrotic cell death induced by beta-lapachone as determined by absence of sub-G1 fraction, externalization of phosphatidylserine. CONCLUSIONS: beta-lapachone induces either apoptotic or necrotic cell death in a variety of human carcinoma cells including ovary, colon, lung, prostate, and breast, suggesting a wide spectrum of anti-cancer activity in vitro. Both apoptotic and necrotic cell death induced by beta-lapachone are preceded by a rapid release of cytochrome C, followed by the activation of caspase 3 in apoptotic cell death but not in necrotic cell death. Our results suggest that beta-lapachone is a potential anti-cancer drug acting on the mitochondrial cytochrome C-caspase pathway, and that cytochrome C is involved in the early phase of necrosis.
Growing evidence exists for a new role for apoptotic cell recognition and clearance in immune homeostasis. Apoptotic cells at all stages, irrespective of membrane integrity, elicit a signature set of signaling events in responding phagocytes, both professional and non-professional. These signaling events are initiated by receptor-mediated recognition of apoptotic determinants, independently of species, cell type, or apoptotic stimulus. We propose that the ability of phagocytes to respond to apoptotic targets with a characteristic set of signaling events comprises a second distinct dimension of innate immunity, as opposed to the traditional innate discrimination of self vs. non-self. We further propose that a loss or abnormality of the signaling events elicited by apoptotic cells, as distinct from the actual clearance of those cells, may predispose to autoimmunity.
PMID: 17516209 CAMSID: cams2362
Innate immunity; apoptosis; autoimmunity; inflammation; phagocytosis; signal transduction
Smooth virulent Brucella abortus strain 2308 (S2308) causes zoonotic brucellosis in cattle and humans. Rough B. abortus strain RB51, derived from S2308, is a live attenuated cattle vaccine strain licensed in the USA and many other countries. Our previous report indicated that RB51, but not S2308, induces a caspase-2-dependent apoptotic and necrotic macrophage cell death. Dendritic cells (DCs) are professional antigen presenting cells critical for bridging innate and adaptive immune responses. In contrast to Brucella-infected macrophages, here we report that S2308 induced higher levels of apoptotic and necrotic cell death in wild type bone marrow-derived DCs (WT BMDCs) than RB51. The RB51 and S2308-induced BMDC cell death was regulated by caspase-2, indicated by the minimal cell death in RB51 and S2308-infected BMDCs isolated from caspase-2 knockout mice (Casp2KO BMDCs). More S2308 bacteria were taken up by Casp2KO BMDCs than wild type BMDCs. Higher levels of S2308 and RB51 cells were found in infected Casp2KO BMDCs compared to infected WT BMDCs at different time points. RB51-infected wild type BMDCs were mature and activated as shown by significantly up-regulated expression of CD40, CD80, CD86, MHC-I, and MHC-II. RB51 induced the production of cytokines TNF-α, IL-6, IFN-γ and IL12/IL23p40 in infected BMDCs. RB51-infected WT BMDCs also stimulated the proliferation of CD4+ and CD8+ T cells compared to uninfected WT BMDCs. However, the maturation, activation, and cytokine secretion are significantly impaired in Casp2KO BMDCs infected with RB51 or Salmonella (control). S2308-infected WT and Casp2KO BMDCs were not activated and could not induce cytokine production. These results demonstrated that virulent smooth strain S2308 induced more apoptotic and necrotic dendritic cell death than live attenuated rough vaccine strain RB51; however, RB51, but not its parent strain S2308, induced caspase-2-mediated DC maturation, cytokine production, antigen presentation, and T cell priming.