More in expectation than in hope: a new attitude to training in clinical academic medicine.
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Attitude↗
Biomedical subjects
J Savill
Publications and source records attributed to J Savill.
Constitutive death of platelets leading to scavenger receptor-mediated phagocytosis. A caspase-independent cell clearance program.
Apoptosis is a physiological program for the deletion of cells in which caspases govern events leading to safe clearance by phagocytes. However, a growing weight of evidence now suggests that not all forms of programmed cell death are caspase-dependent. We now report a complete and constitutive but caspase-independent program for the specific phagocytic clearance of intact effete platelets, anucleated blood cells of critical importance in health and disease. Platelets aged in vitro not only exhibited increased expression of proapoptotic Bak and Bax but also evidenced constitutive diminution of function such as decreased aggregation to ADP, which was accelerated by culture in the absence of plasma. This abrogation of cell function in plasma-deprived platelets was associated with morphological and biochemical features similar to those of granulocyte apoptosis, that is, cytoplasmic condensation, plasma membrane changes including exposure of phosphatidylserine and the granule protein P-selectin, and recognition by phagocyte scavenger receptors. However, and in contrast with constitutive death of other inflammatory blood cells by apoptosis, these events were not affected by caspase inhibitors, nor was there evidence of caspase-3 activation either by hydrolysis of analog peptide substrates or Western blot analysis, serving to emphasize that neither programmed cell death nor clearance by phagocytes need involve caspases.
Apoptosis↗
Glucocorticoids promote nonphlogistic phagocytosis of apoptotic leukocytes.
Phagocyte recognition, uptake, and nonphlogistic degradation of neutrophils and other leukocytes undergoing apoptosis promote the resolution of inflammation. This study assessed the effects of anti-inflammatory glucocorticoids on this leukocyte clearance mechanism. Pretreatment of "semimature" 5-day human monocyte-derived macrophages (M phi) for 24 h with methylprednisolone, dexamethasone, and hydrocortisone, but not the nonglucocorticoid steroids aldosterone, estradiol, and progesterone, potentiated phagocytosis of apoptotic neutrophils. These effects were specific in that the potentiated phagocytosis of apoptotic neutrophils was completely blocked by the glucocorticoid receptor antagonist RU38486, and glucocorticoids did not promote 5-day M phi ingestion of opsonized erythrocytes. Similar glucocorticoid-mediated potentiation was observed with 5-day M phi uptake of alternative apoptotic "targets" (eosinophils and Jurkat T cells) and in uptake of apoptotic neutrophils by alternative phagocytes (human glomerular mesangial cells and murine M phi elicited into the peritoneum or derived from bone marrow). Importantly, methylprednisolone-mediated enhancement of the uptake of apoptotic neutrophils did not trigger the release of the chemokines IL-8 and monocyte chemoattractant protein-1. Furthermore, longer-term potentiation by methylprednisolone was observed in maturing human monocyte-derived M phi, with greater increases in 5-day M phi uptake of apoptotic cells being observed the earlier glucocorticoids were added during monocyte maturation into M phi. We conclude that potentiation of nonphlogistic clearance of apoptotic leukocytes by phagocytes is a hitherto unrecognized property of glucocorticoids that has potential implications for therapies aimed at promoting the resolution of inflammatory diseases.
Animals↗
Phagocytosis triggers macrophage release of Fas ligand and induces apoptosis of bystander leukocytes.
Human monocyte/macrophages (Mphi) exposed to nonparticulate stimuli can express cell surface Fas ligand (FasL) and release active soluble FasL (sFasL). We now report that monocyte/Mphi-ingesting opsonized zymosan released sFasL and conditioned supernatants so that these triggered Fas-mediated apoptosis of "bystander" monocytes and FasL-negative neutrophils. Furthermore, identical results were seen with Mphi taking up apoptotic neutrophils, whereas medium conditioned by Mphi phagocytizing latex beads had no proapoptotic effects upon neutrophils despite the presence of sFasL. These data suggest the hitherto unrecognized existence of a feedback loop requiring soluble factors in addition to sFasL that may promote resolution of inflammation-phagocytic clearance of apoptotic cells leading to Fas-mediated killing of bystander leukocytes by phagocytizing macrophages.
Apoptosis↗
Type IV collagen and laminin regulate glomerular mesangial cell susceptibility to apoptosis via beta(1) integrin-mediated survival signals.
Postinflammatory scarring is characterized by changes in extracellular matrix (ECM) composition and progressive loss of normal resident cells. In glomerular inflammation there is now evidence that unscheduled apoptosis (programmed cell death) of mesangial and other resident cells may mediate progression to irreversible glomerulosclerosis. In the current study we examined the hypothesis that ECM components may differ in their capacity to support mesangial cell survival by suppression of apoptosis. Using a well-established in vitro model of mesangial cell apoptosis, we found that collagen IV and laminin, components of normal mesangial ECM, protected rat mesangial cells from apoptosis induced by serum starvation and DNA damage, by a beta(1) integrin-mediated, but arg-gly-asp (RGD)-independent mechanism. In contrast, collagen I, fibronectin, and osteonectin/SPARC, which are overexpressed in diseased glomeruli, failed to promote rat mesangial cell survival. However, the survival-promoting effect of collagen IV and laminin was not associated with changes in cellular levels of apoptosis regulatory proteins of the Bcl-2 family. These experiments demonstrate that glomerular mesangial cell survival is dependent on interactions with ECM and provide insights into potential mechanisms by which resident cell loss may occur during acute inflammation and postinflammatory scarring of the kidney and other organs.
Animals↗
Regulation of glomerular cell number by apoptosis.
While commonly viewed as leading to glomerular scarring and end-stage renal failure, glomerular cell proliferation may be a beneficial response that promotes the injured glomerulus to return to its proper function. This brief review looks at the potentially counterbalancing influences that cause glomerular cells to survive, undergo mitosis, differentiate or die.
Apoptosis↗
Management of hypertension in patients developing end-stage renal failure.
The benefits of treating hypertension include preventing or delaying the progression of chronic renal failure, and reducing the cardiovascular complications of patients with renal disease. We examined how well hypertension had been managed in all 145 patients from a single health district who started dialysis during a 3-year period. Data relating to management of hypertension, including all blood pressure readings, were obtained from their general practice and hospital case notes. The anonymized data were reviewed by two independent assessors against a set of standards based on the British Hypertension Society guidelines. There was close agreement between the assessors. Complete records were obtained in 98.5% of cases. Of the 145 patients, 107 (76.4%) were hypertensive before developing end-stage renal failure. There were departures from standards in all categories of care: 24.3% in detection/diagnosis, 29% in investigation, 22.4% in referring to a nephrologist, and 17% in follow-up. The British Hypertension Society recommended standard for diastolic blood pressure of 90 mmHg was achieved in only 45%. In 32%, the assessors independently concluded that poor blood pressure control might have affected adversely the progression of renal failure. New methods of dealing with these problems are required and possible approaches are discussed.
Adult↗
More than merely academic: the new Academy of Medical Sciences.
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Academies and Institutes↗
The terminal sequence of complement plays an essential role in antibody-mediated renal cell apoptosis.
Mesangial cell (MC) injury is a characteristic feature in the early phase of Thy.1 nephritis. The present study investigates the contribution of complement to MC apoptosis in this experimental model of kidney disease in rats. Thy.1 nephritis was induced by injection of mouse anti-Thy.1 monoclonal antibody (ER4G). To assess the contribution of the terminal sequence of complement on apoptosis, the studies were performed in complement-sufficient PVG/c (PVG/c+) rats and in rats deficient in complement C6 (PVG/c-). Apoptosis was monitored by assessment of the number of condensed nuclei in kidney sections stained with periodic acid-Schiff (PAS) and by the terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) method and expressed as number of apoptotic cells per 50 glomerular cross sections. In the PAS method, 1 h after intravenous injection of ER4G, PVG/c+ rats exhibited 160.9 +/- 49.5 apoptotic cells, whereas PVG/c- rats had only 3.2 +/- 1.4 apoptotic cells. Control rats exhibited 0.9 +/- 0.6 apoptotic cells. These findings were confirmed with the TUNEL method. In PVG/c- rats, a maximum number of 8.8 +/- 3.1 TUNEL-positive (TUNEL+) cells was found at 6 h followed by a decline thereafter. In PVG/c+ rats, apoptosis was associated with deposition of C6 and C5b-9. Restoration of the complement system of PVG/c- rats with purified human C6 resulted in an increase of apoptosis at 1 h after injection of ER4G from minimal numbers to 239.9 +/- 52.4 TUNEL+ cells. These studies appear to indicate for the first time that the terminal sequence of complement is involved in induction of apoptosis.
Analysis of Variance↗
Constitutive apoptosis in human neutrophils requires synergy between calpains and the proteasome downstream of caspases.
Programmed cell death invariably requires the activation of proteolytic cascades that are not yet well defined but are initiated after apical caspase activation. We provide evidence that calpains and the proteasome function synergistically downstream of caspases to assist the constitutive apoptotic program of aging neutrophils, which plays an important role in resolution of inflammatory responses. Inhibitor studies indicated that "tethering" of preapoptotic senescent neutrophils to human macrophages required caspase activity. However, the development of morphological features characteristic of apoptosis, including nuclear morphology, PS exposure, surface protein shedding, and the capacity to be ingested by macrophages, required the downstream action of either calpains or the proteasome. Calpain activities were constitutively active in freshly isolated neutrophils and responsible for rearrangements in the protein composition and structure of the plasmalemmal cytoskeleton as they aged in culture and underwent apoptosis. This included a dissociation of protein(s) from F-actin, a candidate mechanism for increased susceptibility to cleavage, and a loss in immunodetectable alpha-actinin and ezrin, two actin-binding, membrane-anchoring proteins. These results clarify roles for different classes of proteases in a physiologically important form of constitutive apoptosis.
Actinin↗
Apoptosis. Phagocytic docking without shocking.
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Animals↗
Apoptosis: the importance of being eaten.
In vivo, cells undergoing apoptosis are usually recognised and swiftly ingested by macrophages or neighbouring cells acting as semi-professional phagocytes. This review debates evidence that the contents of apoptotic cells represent a danger to the organism, being capable of injuring tissue directly or triggering autoimmune responses, concluding that phagocytic clearance of intact apoptotic cells is a safe disposal route. Indeed, new data suggest that, in certain circumstances, phagocytes ingesting apoptotic cells may actively downregulate inflammatory and immune responses. Consequently, increasing evidence that there may be factors capable of perturbing safe clearance of apoptotic cells in vivo suggests that failure of this process may be a hitherto unrecognised pathogenetic factor in inflammatory and autoimmune diseases. New treatments designed to promote safe phagocytic clearance of dying cells can be anticipated, and it may even prove possible to eliminate unwanted cells by inducing appearance of cell surface 'eat me' signals.
Apoptosis↗
Cellular apoptosis and proliferation in experimental renal fibrosis.
BACKGROUND: The progression of chronic renal failure (CRF) is associated with the progressive deletion of renal cells along with the fibrosis of the kidney. We have studied the role of programmed cell death (apoptosis) in the progression of experimental CRF and renal scarring. METHODS: The sub-total (5/6th) nephrectomy (SNx) model of CRF was studied in adult male Wistar rats, with renal tissue collected from experimental and control animals on days 7, 15, 30, 60, 90, and 120 post SNx (n = 6 per group). These were examined for morphological signs of apoptosis by light and electron microscopy. Further, we stained the nuclear chromatin by the acridine orange fluorescent method and detected signs of DNA cleavage by endonucleases via the principal of TUNEL staining (ApopTag). Rates of cellular proliferation were measured simultaneously by immunohistochemical staining for the proliferating cell nuclear antigen (PCNA). In addition, cell division was monitored by counting of morphologically mitotic motifs detectable by light microscopy. RESULTS: Progressive renal insufficiency associated with glomerulosclerosis and tubulointerstitial fibrosis took place in the majority of SNx rats. In these animals, we noted a marked and progressive increase in the number of apoptotic glomerular, tubular as well as interstitial cells. The most significant apoptotic changes were seen in the tubules of remnant kidneys peaking at day 120 post-SNx. At this stage, the increase in apoptosis compared to controls was 10.33+/-2.67 (M+/-SEM) fold for glomerular cells (P< or =0.006), 26.20+/-4.56 fold for tubular cells (P < 0.0001) and 4.66+/-0.81 fold for interstitial cells (P< or =0.001). Parallel changes in the number of PCNA positive renal cells were observed. Maximal PCNA staining was seen at day 120 when the increase with respect to controls was 14.00+/-4.93 fold (P< or = 0.05) for glomerular cells, 60.01+/-12.20 fold (P< or =0.05) for tubular cells and 28.59+/-4.45 fold (P< or = 0.05) for interstitial cells. As expected, the number of cells undergoing division and detectable by conventional light microscopy was lower at any time point to those expressing PCNA. We also observed a close correlation between the severity of tubular atrophy and tubulointerstitial fibrosis with the rate of tubular apoptosis (r=0.970, R2 =0.941, P< or =0.001). CONCLUSIONS: We have shown a time-dependent increase in apoptosis and PCNA antigen positive staining in the sub-total nephrectomy model of chronic renal failure correlating with the progression of renal fibrosis. PCNA staining did not match analysis for mitosis and was considered to overestimate the number of proliferating cells in the tissue. With this reservation in mind and taking into account the relative time-frames in vivo of apoptosis and proliferation; apoptosis potentially outweighs proliferation by a factor of 2 8-fold, when examined over the same time period. Consequently, even small changes in the finite numbers of apoptotic cells become highly significant. Our results have shown the definite role of apoptosis within progression of renal damage and highlighted how it may contribute to the progression of tubular atrophy and play a role in the pathogenesis of tubulo-interstitial scarring.
Animals↗
Cytokines promote glomerular mesangial cell survival in vitro by stimulus-dependent inhibition of apoptosis.
Resolution of glomerular inflammation requires the removal of proliferating resident glomerular mesangial cells, but excessive loss of glomerular cells is a feature of postinflammatory scarring. Because apoptosis regulates mesangial cell number in glomerular inflammation, we have studied the exogenous control of apoptosis triggered in cultured mesangial cells by stimuli likely to be important in vivo. Apoptosis could be induced by serum deprivation to model decreased availability of survival factors, by etoposide as an example of DNA-damaging agents, by ligation of mesangial cell Fas, and by protein synthesis inhibition by cycloheximide. Insulin-like growth factor I (IGF-I), IGF-II, and basic fibroblast growth factor were each able to suppress apoptosis induced by serum deprivation, whereas TGF-beta 1, epidermal growth factor, and platelet-derived growth factor had no effect. IGF-I and IGF-II (but not basic fibroblast growth factor) were also able to protect cells from apoptosis induced by etoposide or cycloheximide. However, Fas-mediated apoptosis was resistant to suppression by all three cytokines. None of the cytokines tested caused a change in the levels of expression of Bcl-2, Bax, Bcl-x, or Bak proteins. The survival-promoting properties of serum-free medium conditioned by mesangial cells was abrogated by neutralizing IGF-I Ab. These experiments are the first to define cytokines that inhibit apoptosis and thereby promote survival of mesangial cells, and the data indicate a paracrine survival signaling role for IGF-I. Finally, the data show that Fas ligation can override cytokine survival signaling, emphasizing a candidate role for this molecule in the undesirable apoptotic loss of mesangial cells during the progression of glomerular scarring.
Animals↗
Country profile: United Kingdom.
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Humans↗
Human glomerular mesangial cell phagocytosis of apoptotic neutrophils: mediation by a novel CD36-independent vitronectin receptor/thrombospondin recognition mechanism that is uncoupled from chemokine secretion.
In this study we examined the mechanisms by which glomerular mesangial cells ingest apoptotic cells and the mesangial cell response to this event, since there is in vivo evidence that such semiprofessional phagocytes participate in phagocytic clearance of both apoptotic leukocytes and apoptotic resident cells from inflamed glomeruli, thereby promoting resolution of glomerulonephritis. Mesangial cell phagocytosis of apoptotic neutrophils in vitro was not affected by inhibitors of lectin-like receptors, phosphatidylserine receptors, the 61D3 Ag, and beta1 and beta2 integrins, receptors which have been implicated in phagocytosis of apoptotic cells by particular populations of semiprofessional and professional phagocytes. However, the specific inhibitory effects of cationic aminosugars, Arg-Gly-Asp-Ser (RGDS) peptide, and mAbs to phagocyte alpha(v)beta3 vitronectin receptor integrin and "bridging" thrombospondin 1 (TSP1) indicated that mesangial cell phagocytosis of apoptotic cells involved an alpha(v)beta3/TSP mechanism akin to that described for human monocyte-derived macrophages (Mphi) in which Mphi CD36 plays an important role in binding "bridging" TSP1. However, mesangial cells did not express CD36 and there was no evidence for involvement of alternative phagocyte receptors for TSP1, heparan sulfate proteoglycan and sulfatides. Nevertheless, phagocytosis of apoptotic neutrophils by either mesangial cells or Mphi failed to elicit secretion of IL-8 and MCP-1, representatives of each major class of proinflammatory chemotactic cytokines. We conclude that mesangial cell phagocytosis of apoptotic neutrophils involves a novel CD36-independent, alpha(v)beta3/TSP-mediated mechanism that is uncoupled from chemokine secretion, emphasizing the injury-limiting potential of mesangial cell phagocytosis of apoptotic cells.
Apoptosis↗
Actin is cleaved during constitutive apoptosis.
Proteases play an important role in the programme of cell death by apoptosis but little is known of the substrates cleaved, particularly in constitutive models of this type of cell death. Neutrophils spontaneously undergo apoptosis in culture without requiring external stimuli. During this process we found biochemical and immunochemical evidence for the cleavage of membrane-associated actin, a component of the cytoskeleton that links polymerized actin to the plasma membrane. Cleavage occurred at a single site at the N-terminus, between residues Val43-Met44, a site devoid of a consensus motif for cleavage by cysteine proteases of the interleukin-1beta-converting enzyme (ICE)-family. Whereas actin cleavage and nuclear/cell surface markers of apoptosis were co-ordinately diminished by zVAD-fmk, an inhibitor of the ICE-like family of proteases, only acetyl-leucyl-leucylnormethional, an inhibitor of calpains, was capable of completely inhibiting actin cleavage. Our results suggest that actin is not a direct substrate for the ICE-like family of proteases. By disabling the cytoskeleton, actin cleavage may be an important component in the capacity of apoptosis to reduce the injurious potential of neutrophils.
Actins↗
Role of molecular cell biology in understanding disease.
Molecular techniques have revolutionised our knowledge of cell and tissue function in both health and disease. We already have new and powerful treatments based on an understanding of communication between cells by messenger molecules called cytokines. Furthermore, there is great therapeutic promise in defining molecules which regulate cell adhesion, motility, proliferation, survival, and death. Rational manipulation of cell and tissue function for therapeutic ends may be much closer than you think.
Cell Adhesion Molecules↗