John Savill

A blast from the past: clearance of apoptotic cells regulates immune responses

Apoptosis, which is a programmed and physiological form of cell death, is known to shape the immune system by regulating populations of effector lymphocytes. However, the binding and ingestion of dying cells by monocytes/macrophages and dendritic cells can also influence immune responses markedly by enhancing or suppressing inflammation. Therefore, dead cells, which are a reflection of an organisms immediate past, can control its immunological future.

Apoptosis in resolution of inflammation.

The last few years have seen the accumulation of compelling evidence that apoptosis (programmed cell death) plays a major role in promoting resolution of the acute inflammatory response. Neutrophils are constitutively programmed to undergo apoptosis, which limits their pro‐inflammatory potential and leads to rapid, specific, and non‐phlogistic recognition by macrophages and semi‐professional phagocytes. Similar mechanisms have been implicated in clearance of eosinophils, lymphocytes, and monocytes and apoptosis also plays a role in remodeling the inflamed site by deletion of myofibroblasts. A growing understanding of the mechanisms regulating leukocyte apoptosis and of the molecules mediating safe phagocytic clearance of dying cells may yield new insights into the pathogenesis and therapy of inflammatory diseases. J. Leukoc. Biol. 61: 375–380; 1997.

Apoptosis disables CD31-mediated cell detachment from phagocytes promoting binding and engulfment

Macrophage recognition and ingestion of ‘self’ cells undergoing apoptosis in vivo protects tissues from the toxic contents of dying cells and modulates macrophage regulation of inflammatory and immune responses. However, the complex molecular mechanisms mediating macrophage discrimination between viable and apoptotic cells are poorly understood. In particular, little is known of why viable nucleated cells are not engulfed by macrophages. To reveal active repulsion of viable cells and to seek specific capture or ‘tethering’ of apoptotic cells, we studied macrophage binding of viable and apoptotic leukocytes under conditions of flow. We found that homophilic ligation of CD31 (ref. 4) on viable leukocytes promoted their active, temperature-dependent detachment under low shear, whereas such CD31-mediated detachment was disabled in apoptotic leukocytes, promoting tight binding and macrophage ingestion of dying cells. Here we propose that CD31 (also known as platelet-endothelial cell adhesion molecule-1, PECAM-1) is an example of a cell-surface molecule that prevents phagocyte ingestion of closely apposed viable cells by transmitting ‘detachment’ signals, and which changes function on apoptosis, promoting tethering of dying cells to phagocytes.

Selective ablation of αv integrins in the central nervous system leads to cerebral hemorrhage, seizures, axonal degeneration and premature death

Mouse embryos genetically null for all αv integrins develop intracerebral hemorrhage owing to defective interactions between blood vessels and brain parenchymal cells. Here, we have used conditional knockout technology to address whether the cerebral hemorrhage is due to primary defects in vascular or neural cell types. We show that ablating αv expression in the vascular endothelium has no detectable effect on cerebral blood vessel development, whereas deletion of αv expression in central nervous system glial cells leads to embryonic and neonatal cerebral hemorrhage. Conditional deletion of αv integrin in both central nervous system glia and neurons also leads to cerebral hemorrhage, but additionally to severe neurological defects. Approximately 30% of these mutants develop seizures and die by 4 weeks of age. The remaining mutants survive for several months, but develop axonal deterioration in the spinal cord and cerebellum, leading to ataxia and loss of hindlimb coordination. Collectively, these data provide evidence that αv integrins on embryonic central nervous system neural cells, particularly glia, are necessary for proper cerebral blood vessel development, and also reveal a novel function for αv integrins expressed on axons in the postnatal central nervous system.

Apoptotic cells and innate immune stimuli combine to regulate macrophage cytokine secretion.

Macrophage interactions with apoptotic cells can suppress inflammatory responses, but cell death by apoptosis may also trigger inflammation. We now report that murine macrophages exposed to the combination of apoptotic cells and archetypal ligands for Toll-like receptors (TLRs) 2, 4, and 9 mount cytokine responses that differ importantly from those elicited by either class of stimulus alone. TLR ligands induced early and sustained secretion of TNF-α, macrophage-inflammatory protein (MIP) 1α and MIP-2 with later secretion of IL-10, IL-12, and TGF-β1; apoptotic cells alone stimulated late TGF-β1 secretion only. The combination of apoptotic cells and TLR ligands enhanced early secretion of TNF-α, MIP-1α, and MIP-2 and increased late TGF-β1 secretion, while suppressing late TNF-α, IL-10, and Il-12 by mechanisms which could nevertheless be overridden by IFN-γ. We propose that this combinatorial macrophage cytokine response to apoptotic cells and TLR ligands may contribute to recruitment and activation of innate immune defense when cell death occurs at infected inflamed sites while promoting later resolution with diminished engagement of adaptive immunity.

Granulocyte clearance by apoptosis in the resolution ofinflammation

It has recently been recognized that extravasated granulocytes undergo apoptosis or programmed cell death. This process controls the functional longevity of these cells and is exquisitely modulated by environmental inflammatory mediators. By contrast with necrosis, an alternative fate for granulocytes in tissues, during apoptosis the granulocyte membrane remains intact and potentially injurious granule contents are retained. The intact apoptotic cell is removed by macrophages utilizing novel surface recognition mechanisms which fail to trigger a pro-inflammatory macrophage response. The balance between granulocyte apoptosis and necrosis in inflamed tissues may be an important determinant of the degree of tissue injury, and further dissection of the mechanisms of granulocyte apoptosis and removal may lead to new therapeutic strategies in inflammatory disease.

Compartmentalized megakaryocyte death generates functional platelets committed to caspase-independent death

Caspase-directed apoptosis usually fragments cells, releasing nonfunctional, prothrombogenic, membrane-bound apoptotic bodies marked for rapid engulfment by macrophages. Blood platelets are functional anucleate cells generated by specialized fragmentation of their progenitors, megakaryocytes (MKs), but committed to a constitutive caspase-independent death. Constitutive formation of the proplatelet-bearing MK was recently reported to be caspase-dependent, apparently involving mitochondrial release of cytochrome c, a known pro-apoptogenic factor. We extend those studies and report that activation of caspases in MKs, either constitutively or after Fas ligation, yields platelets that are functionally responsive and evade immediate phagocytic clearance, and retain mitochondrial transmembrane potential until constitutive platelet death ensues. Furthermore, the exclusion from the platelet progeny of caspase-9 present in the progenitor accounts for failure of mitochondrial release of cytochrome c to activate caspase-3 during platelet death. Thus, progenitor cell death by apoptosis can result in birth of multiple functional anucleate daughter cells.

Activated macrophages direct apoptosis and suppress mitosis of mesangial cells

During inflammation in the glomerulus, the complement of resident myofibroblast-like mesangial cells is regulated by mitosis and apoptosis, but the cellular mechanisms controlling the size of mesangial cell populations have remained obscure. Prompted by studies of development, we sought evidence that macrophages regulate mesangial cell number. Rat bone marrow-derived macrophages primed with IFN-γ then further activated in coculture with LPS or TNF-α elicited a 10-fold induction of rat mesangial cell apoptosis and complete suppression of mitosis, effects inhibitable by the NO synthase inhibitors l-monomethyl arginine and l-N6-(1-iminoethyl) lysine dihydrochloride. Complete dependence upon macrophage-derived NO was observed in comparable experiments employing activated bone marrow macrophages from wild-type and NO synthase 2−/− mice. Nevertheless, when mesangial cells were primed with IFN-γ plus TNF-α, increased induction by activated macrophages of mesangial apoptosis exhibited a NO-independent element. The use of gld/gld macrophages excluded a role for Fas ligand in this residual kill, despite increased expression of Fas and increased susceptibility to soluble Fas ligand exhibited by cytokine-primed mesangial cells. Finally, activated macrophages isolated from the glomeruli of rats with nephrotoxic nephritis also induced apoptosis and suppressed mitosis in mesangial cells by an l-monomethyl arginine-inhibitable mechanism. These data demonstrate that activated macrophages, via the release of NO and other mediators, regulate mesangial cell populations in vitro and may therefore control the mesangial cell complement at inflamed sites.

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 α-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.

Type IV Collagen and Laminin Regulate Glomerular Mesangial Cell Susceptibility to Apoptosis Via β1 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.