Cell death is a ubiquitous physiological process that serves a critical role in organismal development and in homeostasis. In particular, it plays a pivotal role in shaping and maintaining functional cellular repertoires, including a non-autoreactive and self-limiting immune system and an appropriately interconnected and outwardly targeted neuronal network. The purpose of the physiological cell death process, then, is the elimination of functionally inappropriate cells in a manner that does not elicit a pathological or inflammatory response.
How is orderly and non-inflammatory cellular elimination accomplished? We have focused on a molecular dissection of two critical aspects of the physiological cell death process: the cell-autonomous death effector mechanism, including the point of irreversible death commitment, and the process of corpse recognition leading to non-inflammatory phagocytic clearance.

Deaths In The Lives Of Cells:

While the induction of physiological death in disparate cell types is effected by a wide variety of stimuli, a common pathway of lethality, resulting in a common apoptotic morphology, operates. We find that the critical elements of this death process comprise a conserved and ordered sequence of activities activated post-translationally from resident molecules. Diverse death signals act afferently via idiosyncratic signaling pathways to activate these essential elements, which include a proteolytic cascade of caspase and subsequent cyclin dependent kinase (Cdk) molecules. This finding provides a molecular basis to the long-appreciated and complex linkage between the physiological cell death process and the productive cell cycle, and suggests new approaches to the regulation of cell death responsiveness.

We have tested the functional role of Cdk activity in cell death genetically, with the expression of dominant negative Cdk mutants and Cdk inhibitor proteins. Transient blockade of caspase-dependent Cdk2 activity confers long-lived sparing from death on cells otherwise triggered to die. Susceptibility to the induction of cell death, including the activation of the caspase cascade, is unimpaired in cells spared from death by inhibition of Cdk activity. These data reveal that the action of Cdks downstream of caspases represents a conserved phase of the cell death process, closely linked with actual lethality. Our results suggest that caspase activity is necessary, but not sufficient, for the physiological cell death response and that a requisite function of the proteolytic caspase cascade is the post-translational activation of death-effector Cdks. The identification of caspase-dependent Cdk substrates is a focus of current work.

What Becomes Of The Corpse?

In contrast to the active and ordered physiological (apoptotic) cell death process, pathological (necrotic) cell deaths are chaotic and passive. The distinction between physiological and pathological deaths, in addition to reflecting mechanistic differences in cellular disintegration, is of functional significance with respect to the different outcomes triggered by the corpses. Our studies have revealed that macrophages and other phagocytic cells recognize and engulf these two classes of dead cells via distinct and non-competing mechanisms. Linked to their recognition, apoptotic corpses profoundly modulate the pro-inflammatory responses of phagocytic cells with which they interact. Paradoxically, this represents a gain-of-function acquired during the physiological cell death process. Necrotic cells, in contrast, do not down-regulate macrophage inflammatory responses.

Modulation of inflammatory responsiveness is exerted on the level of the pro-inflammatory transcriptional machinery of phagocytes with which the apoptotic cells interact. Specifically, their effect is an immediate-early inhibition of pro-inflammatory cytokine gene transcription, and is triggered directly upon binding to the macrophage, independent of subsequent engulfment and soluble factor involvement. Apoptotic recognition and inflammatory modulation are key elements of an innate immune response that discriminates live from dead (or effete) cells. This is distinct from the innate immune discrimination of self from other with which Toll-like receptors are involved.

Like other aspects of the definitive cell death process, novel cell surface determinants for their recognition arise post-translationally on apoptotic cells during the cell death process. These determinants are widely conserved among species. We are taking a variety of genomic and proteomic approaches to the identification of these apoptotic ligands for recognition and response modulation. A fuller understanding of the process of immune modulation exerted physiologically by apoptotic cells has enormous potential significance for efforts to intervene in cases of deleterious and pathological inflammatory responses.