Mechanisms of internalization of apoptotic and necrotic L929 cells by a macrophage cell line studied by electron microscopy

Rapid and efficient phagocytic removal of dying cells is a key feature of apoptosis. In necrotic caspase-independent modes of death, the role and extent of phagocytosis is not well documented. To address this issue, we studied at the ultrastructural level the phagocytic response to dying cells in an in vitro phagocytosis assay with a mouse macrophage cell line (Mf4/4). As target cells, murine L929sAhFas cells were induced to die by TNFR1-mediated necrosis or by Fas-mediated apoptosis. Apoptotic L929sAhFas cells are taken up by complete engulfment of apoptotic bodies as single entities forming a tight-fitting phagosome, thus resembling the "zipper"-like mechanism of internalization. In contrast, primary and secondary necrotic cells were internalized by a macropinocytotic mechanism with formation of multiple ruffles by the ingesting macrophage. Ingestion of necrotic cellular material was invariably taking place after the integrity of the cell membrane was lost and did not occur as discrete particles, in contrast to apoptotic material that is surrounded by an intact membrane. Although nuclei of necrotic cells have been observed in the vicinity of macrophages, no uptake of necrotic nuclei was observed. The present report provides a basis for future studies aimed at discovering molecular pathways that precede these diverse mechanisms of uptake.     

Cover Picture

Various tumor cells and normal skin fibroblasts can act as nonprofessional phagocytes (green) that engulf necrotic cells (red). After completed engulfment, the necrotic cell pushes the crescent shaped nucleus of the phagocytic cell towards the periphery. Confocal laser scanning microscopy, blue: DNA.     

Phagocytosis of necrotic cells by macrophages is phosphatidylserine dependent and does not induce inflammatory cytokine production

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.     

Airway Management in Croup and Epiglottitis

Treatment techniques for airway obstruction in croup and epiglottitis are reviewed in the medical literature. Series totaling 295 nasotracheal intubations, and 591 tracheostomies were reviewed. There were two deaths attributable to airway complications in 126 patients in whom nasotracheal intubation was carried out. In three patients subglottic granulation tissue and subglottic stenoses developed from short-term nasotracheal intubation. There were no subglottic stenoses or tracheal stenoses reported in the 591 tracheostomies. From this review, it would seem feasible to use nasotracheal intubation for short-term airway treatment in croup and epiglottitis. The increasing occurrence of laryngeal and tracheal complications with long-term intubation suggests that tracheostomy be considered in such cases.     

Epiglottitis--an increasing problem for adults.

In a 2-year period, 9 adults were admitted to hospital with acute epiglottitis confirmed by direct laryngoscopy or lateral neck radiograph, or both. The mean age was 53 +/- 14 years, with acute epiglottitis occurring in 89% during the months of September to March. Intubation was required in 4 patients. The duration of symptoms was 7.8 +/- 2.4 hours for intubated patients versus 18.8 +/- 8.9 hours for those not intubated. For 6 patients an incorrect diagnosis was made on their first presentation. All 8 patients having laryngoscopy had typical findings, and none had respiratory obstruction precipitated by the procedure. In 5 patients blood cultures were positive, 4 for Hemophilus influenzae type b, and 1 for Streptococcus pneumoniae. In 2 patients the H influenzae was ampicillin-resistant. All patients recovered after receiving parenteral steroid therapy and appropriate antibiotics.     

Necrotic Cells Actively Attract Phagocytes through the Collaborative Action of Two Distinct PS-Exposure Mechanisms

Necrosis, a kind of cell death closely associated with pathogenesis and genetic programs, is distinct from apoptosis in both morphology and mechanism. Like apoptotic cells, necrotic cells are swiftly removed from animal bodies to prevent harmful inflammatory and autoimmune responses. In the nematode Caenorhabditis elegans, gain-of-function mutations in certain ion channel subunits result in the excitotoxic necrosis of six touch neurons and their subsequent engulfment and degradation inside engulfing cells. How necrotic cells are recognized by engulfing cells is unclear. Phosphatidylserine (PS) is an important apoptotic-cell surface signal that attracts engulfing cells. Here we observed PS exposure on the surface of necrotic touch neurons. In addition, the phagocytic receptor CED-1 clusters around necrotic cells and promotes their engulfment. The extracellular domain of CED-1 associates with PS in vitro. We further identified a necrotic cell-specific function of CED-7, a member of the ATP-binding cassette (ABC) transporter family, in promoting PS exposure. In addition to CED-7, anoctamin homolog-1 (ANOH-1), the C. elegans homolog of the mammalian Ca²⁺-dependent phospholipid scramblase TMEM16F, plays an independent role in promoting PS exposure on necrotic cells. The combined activities from CED-7 and ANOH-1 ensure efficient exposure of PS on necrotic cells to attract their phagocytes. In addition, CED-8, the C. elegans homolog of mammalian Xk-related protein 8 also makes a contribution to necrotic cell-removal at the first larval stage. Our work indicates that cells killed by different mechanisms (necrosis or apoptosis) expose a common “eat me” signal to attract their phagocytic receptor(s); furthermore, unlike what was previously believed, necrotic cells actively present PS on their outer surfaces through at least two distinct molecular mechanisms rather than leaking out PS passively.     

Histidine-rich glycoprotein specifically binds to necrotic cells via its amino-terminal domain and facilitates necrotic cell phagocytosis

Cells that become necrotic or apoptotic through tissue damage or during normal cellular turnover are usually rapidly cleared from the circulation and tissues by phagocytic cells. A number of soluble proteins have been identified that facilitate the phagocytosis of apoptotic cells, but few proteins have been defined that selectively opsonize necrotic cells. Previous studies have shown that histidine-rich glycoprotein (HRG), an abundant (approximately 100 microg/ml) 75-kDa plasma glycoprotein, binds to cell surface heparan sulfate on viable cells and cross-links other ligands, such as plasminogen, to the cell surface. In this study we have demonstrated that HRG also binds very strongly, in a heparan sulfate-independent manner, to cytoplasmic ligand(s) exposed in necrotic cells. This interaction is mediated by the amino-terminal domain of HRG and results in enhanced phagocytosis of the necrotic cells by a monocytic cell line. In contrast, it was found that HRG binds poorly to and does not opsonize early stage apoptotic cells. Thus, HRG has the unique property of selectively recognizing necrotic cells and may play an important physiological role in vivo by facilitating the uptake and clearance of necrotic, but not apoptotic, cells by phagocytes.     

The immune reaction against allogeneic necrotic cells is reduced in Annexin A5 knock out mice whose macrophages display an anti-inflammatory phenotype

Proteins of the annexin family bind to phospholipids in a Ca²⁺ dependent manner. The exposure of phosphatidylserine (PS) by apoptotic as well as necrotic cells is one major eat-me-signal for macrophages. Annexin A5 (Anx A5) preferentially binds to PS. The availability of Anx A5 knock out (KO) mice allowed us to investigate for the first time if endogenous Anx A5 modulates the immune response towards allogeneic cells. Furthermore, the effect of Anx A5 gene deletion on the phagocytic process as well as on the inflammatory reaction of macrophages was explored. We found that Anx A5 KO mice have a strongly reduced allogeneic cellular immune reaction against primary as well as secondary necrotic cells. In vivo phagocytosis experiments revealed that macrophages of Anx A5 KO mice displayed an increased uptake of necrotic cells. Additionally, an increased secretion of the anti-inflammatory cytokine IL-10 of isolated macrophages of Anx A5 KO mice after contact with necrotic cells was observed. Furthermore, the promoter activity of the Anx A5 gene was enhanced after stimulation of macrophages. The tumour size of an allogeneic tumour regressed faster when endogenous Anx A5 was present. These data demonstrate that endogenous Anx A5 influences the phagocytosis of necrotic cells, modulates the immune response towards allogeneic cells and acts as an inflammatory protein.     

Detecting breast cancer

A 7-year-old boy, diagnosed as having croup, develops an upper airway obstruction due to epiglottitis during the therapy, resulting in cerebral anoxia. Pediatricians to whom the boy is referred feel that failure to consider epiglottitis in the original diagnosis constitutes negligence. The parents suspect nothing. What should the pediatricians say or do?     

Beware of thermal epiglottis! A case report describing ‘teapot syndrome’

Background

The type of scalding injury known as ‘teapot syndrome’, where hot liquid is grabbed by the child with the aim of ingestion and falls over a child causing burns on the face, upper thorax and arms, is known to cause peri-oral and facial oedema. Thermal epiglottitis following scalds to face, neck and thorax is rare and can occur even in absence of ingestion of a damaging agent or intraoral burns, Awareness of the possibility of thermal epiglottitis, also in scald burns, is imperative to ensure prompt airway protection.

Case presentation

We report the case of a child with thermal epiglottitis after a scalding burn from boiling milk resulting in mixed deep burns of the face, neck and chest, but no history of ingestion. Upon presentation there was a progressive stridor and signs of respiratory distress requiring intubation. Laryngoscopy revealed epiglottis oedema, confirming the diagnosis of thermal epiglottitis. Final extubation took place 5 days after initial burn.

Conclusions

Thermal epiglottitis following scalds to face, neck and thorax is rare and can occur even in absence of ingestion and intra-oral damage. Burns to the peri-oral area should raise suspicion of additional damage to oral cavity and supraglottic structures, even in absence of intra-oral injury or initial respiratory distress. Awareness of the occurrence of thermal epiglottitis in absence of intra-oral injury is important to diagnose impending upper airway obstruction requiring intubation.

     

Identification of the Low Density Lipoprotein (LDL) Receptor-related Protein-1 Interactome in Central Nervous System Myelin Suggests a Role in the Clearance of Necrotic Cell Debris

In the central nervous system (CNS), fast neuronal signals are facilitated by the oligodendrocyte-produced myelin sheath. Oligodendrocyte turnover or injury generates myelin debris that is usually promptly cleared by phagocytic cells. Failure to remove dying oligodendrocytes leads to accumulation of degraded myelin, which, if recognized by the immune system, may contribute to the development of autoimmunity in diseases such as multiple sclerosis. We recently identified low density lipoprotein receptor-related protein-1 (LRP1) as a novel phagocytic receptor for myelin debris. Here, we report characterization of the LRP1 interactome in CNS myelin. Fusion proteins were designed corresponding to the extracellular ligand-binding domains of LRP1. LRP1 partners were isolated by affinity purification and characterized by mass spectrometry. We report that LRP1 binds intracellular proteins via its extracellular domain and functions as a receptor for necrotic cells. Peptidyl arginine deiminase-2 and cyclic nucleotide phosphodiesterase are novel LRP1 ligands identified in our screen, which interact with full-length LRP1. Furthermore, the extracellular domain of LRP1 is a target of peptidyl arginine deiminase-2-mediated deimination in vitro. We propose that LRP1 functions as a receptor for endocytosis of intracellular components released during cellular damage and necrosis.     

Recognition of Apoptotic Cells by Epithelial Cells: CONSERVED VERSUS TISSUE-SPECIFIC SIGNALING RESPONSES*

During apoptosis, cells acquire new activities that enable them to modulate the fate and function of interacting phagocytes, particularly macrophages (mϕ). Although the best known of these activities is anti-inflammatory, apoptotic targets also influence mϕ survival and proliferation by modulating proximal signaling events, such as MAPK modules and Akt. We asked whether modulation of these same signaling events extends to epithelial cells, a minimally phagocytic cell type. We used BU.MPT cells, a mouse kidney epithelial cell line, as our primary model, but we also evaluated several epithelial cell lines of distinct tissue origins. Like mϕ, mouse kidney epithelial cells recognized apoptotic and necrotic targets through distinct non-competing receptors, albeit with lower binding capacity and markedly reduced phagocytosis. Also, modulation of inflammatory activity and MAPK-dependent signaling by apoptotic and necrotic targets was indistinguishable in kidney epithelial cells and mϕ. In contrast, modulation of Akt-dependent signaling differed dramatically between kidney epithelial cells and mϕ. In kidney epithelial cells, modulation of Akt was linked to target cell recognition, independently of phagocytosis, whereas in mϕ, modulation was linked to phagocytosis. Moreover, recognition of apoptotic and necrotic targets by kidney epithelial cells elicited opposite responses; apoptotic targets inhibited whereas necrotic targets stimulated Akt activity. These data confirm that nonprofessional phagocytes recognize and respond to dying cells, albeit in a manner partially distinct from mϕ. By acting as sentinels of environmental change, apoptotic and necrotic targets may permit neighboring viable cells, especially non-migratory epithelial cells, to monitor and adapt to local stresses.     

Phagocytic activity of neutrophils from the peritoneal dialysate of patients with chronic renal failure treated by intermittent peritoneal dialysis.

The phagocytic activity of peritoneal neutrophils was assessed using Bacto-Latex in 50 patients with chronic renal failure treated with intermittent peritoneal dialysis, and in 30 control patients with normal renal function. In the group of patients treated with peritoneal dialysis 20 were additionally investigated while developing peritonitis. A significant decrease in the phagocytic activity of neutrophils was observed in the both dialysed groups, as compared with control subjects. Moreover, the phagocytic activity was significantly lower in patients with peritonitis as compared with dialysed patients without this complication.     

ACUTE EPIGLOTTITIS

Although acute epiglottitis is not a rare disease and may be very severe or fatal, it is one not familiar, as it should be, to all physicians dealing with children.Diagnosis may be confirmed clinically by direct or indirect examination of the epiglottis. Vaporized cool water is preferable to steam for reducing the swelling of mucosal tissues that impairs breathing.Chloramphenicol is the drug of choice, as the majority of cases of acute epiglottitis are due to H. Influenzae.Tracheotomy must be carried out if necessary to maintain an airway.     

Automation and computerization of chemiluminescence: A new methodological approach in the study of human phagocytes

Peripheral blood phagocytic cells (PMNLs) are activated by contact with opsonized particles. Metabolic activation of PMNLs is associated with a remarkable increase in the respiratory burst and generates high energy oxygen compounds which are responsible for the bactericidal activity of PMNLs and for their ability to produce luminol-dependent chemiluminescence (CL). The CL phenomenon is measured by an automated and computerized photoluminometer (Berthold LB950) in whole blood stimulated with opsonized zymosan. This whole blood method of CL measurement has been applied to the study of the phagocytic process and to the investigation of cellular and humoral abnormalities in several pathologies, indicating this assay as a simple, rapid and reliable test.     

Influence of HIV-infection on the phagocytic activity of monocytes/macrophages and granulocytes

Because of the great importance of phagocytosis as a key process in host defence, the influence of HIV-infection on the phagocytic activity of monocytes/macrophages (M0/MAC) and granulocytes was investigated. Therefore, blood samples from the peripheral blood of 70 HIV-infected individuals were incubated with fluorescein isothiocyanate (FITC) labeled Escherichia coli. The uptake of the bacteria was monitored by flow cytometer analysis. A strong and significant increase in the relative number of phagocytic granulocytes was observed ranging from 12.8% in an uninfected control collective to over 30% in AIDS patients. This effect was obtained for all patients and independent of the stage of disease. For monocytes, only marginal changes were found in their phagocytic function. These data suggest that the high susceptibility of HIV patients for secondary infections is not linked to a loss of phagocytic ability of monocytes/macrophages and/or granulocytes.     

Phagocytic capacity of human leukocytes preincubated with Imuran and prednisone

The phagocytic and microbicidal activities of human polymorphonuclear leukocytes (PMNL) were tested after a short-term preincubation of phagocytic cells with Imuran or Prednisone, using C. albicans strains as a test organism. The tests showed that both immunosuppressive agents reduced significantly the microbicidal activity of PMNL cells by 35-40%. Detectable differences in the phagocytic activity and phagocytic index values were not statistically significant. The decreased ability of PMNLs to kill ingested C. albicans strains appears thus to sensitively reflect the altered PMNL function and can be utilized in the biologic monitoring of immunosuppression. The reduced microbicidal potential of phagocytic cells may be also one of the causes of recurrent microbial infections that are so frequent in immunosuppressant-treated patients.     

An unusual clinical and pathological variant of malignant catarrhal fever in a white-tailed deer

A captive male white-tailed deer (Odocoileus virginianus) developed an acute illness over a 3-day period characterized predominantly by neurological, ocular and respiratory signs which were accompanied by prominent gross lesions of multiple organ systems. Histologically, a proliferative vasculitis consisting primarily of lymphocytic-lymphoblastic cellular infiltration was found in ocular, oral, respiratory, cardiac and neural tissues. The extensive nature of these infiltrations resulted in grossly apparent nodular foci in the lung, lymphoid tissue and myocardium which were suggestive of a lymphoproliferative disorder. This is contrasted to the more necrotizing nature of the vasculitis observed in other reported cases of malignant catarrhal fever in white-tailed deer. Although virus isolation was not attempted, serologic findings of antibodies to malignant catarrhal fever virus detected by indirect immunofluorescence and virus neutralization supported a diagnosis of malignant catarrhal fever in this deer.     

Mechanisms of failed apoptotic cell clearance by phagocyte subsets in cardiovascular disease

Recent evidence in humans indicate that defective phagocytic clearance of dying cells is linked to progression of advanced atherosclerotic lesions, the precursor to atherothrombosis, ischemic heart disease, and leading cause of death in the industrialized world. During atherogenesis, apoptotic cell turnover in the vascular wall is counterbalanced by neighboring phagocytes with high clearance efficiency, thereby limiting cellularity and maintaining lesion integrity. However, as lesions mature, phagocytic removal of apoptotic cells (efferocytosis) becomes defective, leading to secondary necrosis, expansion of plaque necrotic cores, and susceptibility to rupture. Recent genetic causation studies in experimental rodents have implicated key molecular regulators of efferocytosis in atherosclerotic progression. These include MER tyrosine kinase (MERTK), milk fat globule-EGF factor 8 (MFGE8), and complement C1q. At the cellular level, atheromata are infiltrated by a heterogenous population of professional phagocytes, comprised of monocytes, differentiated macrophages, and CD11c⁺ dendritic-like cells. Each cell type is characterized by disparate clearance efficiencies and varying activities of key phagocytic signaling molecules. It is in this context that we outline a working model whereby plaque necrosis and destabilization is jointly promoted by (1) direct inhibition of core phagocytic signaling pathways and (2) expansion of phagocyte subsets with poor clearance capacity. Towards identifying targets for promoting efficient apoptotic cell clearance and resolving inflammation in atherosclerosis and during ischemic heart disease and post myocardial infarction, this review will discuss potential in vivo suppressors of efferocytosis at each stage of clearance and how these putative interventional targets may differentially affect uptake at the level of vascular phagocyte subsets.     

Occurrence of herpes simplex virus 1 and three periodontal bacteria in patients with chronic periodontitis and necrotic pulp

Viral and bacterial associations appear to be implicated in the development of periodontal infections. Little information is available describing the periodontopathic agents in root canals with necrotic pulp. In this study, the occurrence and the combinations among herpes simplex virus type 1 (HSV-1) and Dialister pneumosintes, Tannerella forsythia, and Treponema denticola in patients with chronic periodontitis and necrotic pulp were evaluated. Clinical samples from healthy subjects and patients with periodontal or pulp infections were analyzed using a nested polymerase chain reaction PCR to detect HSV and PCR to detect the 3 periodontal bacteria. The presence of Tannerella forsythia and Treponema denticola was observed in healthy, periodontitis, and necrotic pulp patients. HSV was observed in periodontitis and necrotic pulp patients, and no healthy subject harbored D. pneumosintes or HSV. The occurrence of Tannerella forsythia was not statistically significant in patients with necrotic pulp (P = 0.704). Periodontal bacteria were observed varying from 10.3% to 20.7% in periodontitis and necrotic pulp patients. The presence of Treponema denticola - HSV association was predominant in patients showing necrotic pulp (24.1%); however, HSV alone was observed in one patient with periodontitis and in another patient with necrotic pulp. The presence of double association among bacteria or bacteria - HSV could indicate a role in both periodontitis and necrotic pulp, and Tannerella forsythia - Treponema denticola - HSV and Tannerella forsythia - D. pneumosintes - Treponema denticola - HSV associations might be important in periodontitis.