David S. Ucker

Mesenchymal stem cells suppress lymphocyte proliferation in vitro and prolong skin graft survival in vivo

OBJECTIVE Mesenchymal stem cells (MSCs), multipotential cells that reside within the bone marrow, can be induced to differentiate into various components of the marrow microenvironment, such as bone, adipose, and stromal tissues. The bone marrow microenvironment is vital to the development, differentiation, and regulation of the lymphohematopoietic system. We hypothesized that the activities of MSCs in the bone marrow microenvironment might also include immunomodulatory effects on lymphocytes. METHODS Baboon MSCs were tested in vitro for their ability to elicit a proliferative response from allogeneic lymphocytes, to inhibit an ongoing allogeneic response, and to inhibit a proliferative response to potent T-cell mitogens. In vivo effects were tested by intravenous administration of donor MSCs to MHC-mismatched recipient baboons prior to placement of autologous, donor, and third-party skin grafts. RESULTS MSCs failed to elicit a proliferative response from allogeneic lymphocytes. MSCs added into a mixed lymphocyte reaction, either on day 0 or on day 3, or to mitogen-stimulated lymphocytes, led to a greater than 50% reduction in proliferative activity. This effect could be maximized by escalating the dose of MSCs and could be reduced with the addition of exogenous IL-2. In vivo administration of MSCs led to prolonged skin graft survival when compared to control animals: 11.3 +/- 0.3 vs 7 +/- 0. CONCLUSIONS Baboon MSCs have been observed to alter lymphocyte reactivity to allogeneic target cells and tissues. These immunoregulatory features may prove useful in future applications of tissue regeneration and stem cell engineering.

Innate Immune Discrimination of Apoptotic Cells: Repression of Proinflammatory Macrophage Transcription Is Coupled Directly to Specific Recognition

Physiological cell death is a process the purpose of which is the elimination of functionally inappropriate cells in a manner that does not elicit an inflammatory response. We have shown previously that the ability of apoptotic corpses to be recognized by macrophages and to modulate the proinflammatory responses of those cells represents paradoxically a gain-of-function acquired during the physiological cell death process. Cells that die pathologically (that is, necrotic vs apoptotic corpses) also are recognized by macrophages but do not down-regulate macrophage inflammatory responses; the recognition of these two classes of native dying cells occurs via distinct and noncompeting mechanisms. We have examined the apoptotic modulation of proinflammatory cytokine gene transcription in macrophages (by real-time RT-PCR analysis) and the corresponding modulation of transcriptional activators (by transcriptional reporter analyses). Our data demonstrate that apoptotic cells target the proinflammatory transcriptional machinery of macrophages with which they interact, without apparent effect on proximal steps of Toll-like receptor signaling. The modulatory activity of the corpse is manifest as an immediate-early inhibition of proinflammatory cytokine gene transcription, and is exerted directly upon binding to the macrophage, independent of subsequent engulfment and soluble factor involvement. Recognition and inflammatory modulation represent key elements of an innate immune response that discriminates live from effete cells, and without regard to self.

Apoptotic cells, at all stages of the death process, trigger characteristic signaling events that are divergent from and dominant over those triggered by necrotic cells: Implications for the delayed clearance model of autoimmunity

Current models of autoimmunity suggest that delayed clearance of apoptotic cells leads to the presentation of apoptotic antigens in the context of inflammatory signals, with resultant autoimmunity. These models implicitly assume that, in contrast to early apoptotic cells (that retain membrane integrity), late apoptotic cells (with compromised membranes) act like necrotic cells (which also lack intact membranes), possibly because of the release of proinflammatory intracellular contents. We showed previously that early apoptotic and necrotic cells induce distinct mitogen-activated protein kinase modules in macrophages with which they interact. Exposure to apoptotic cells led to nearly complete inhibition of both basal and macrophage colony-stimulating factor-induced ERK1/2 by macrophages. In contrast, necrotic cells induced ERK1/2. We show here that apoptotic cells also strongly induced both c-Jun N-terminal kinase and p38, whereas necrotic cells had no detectable effect on c-Jun N-terminal kinase and p38. We also compared the signaling events induced in macrophages by exposure to early apoptotic cells, late apoptotic cells, and necrotic cells. The signaling events induced by late apoptotic cells were identical to and just as potent as those induced by early apoptotic cells. Thus, apoptotic cells are functionally equivalent throughout the cell death process, irrespective of membrane integrity. Moreover, the effects of both early and late apoptotic cells on signaling were dominant over those of necrotic cells. These data show that apoptotic cells do not become proinflammatory upon the loss of membrane integrity and are inconsistent with the notion that delayed clearance alone can lead to autoimmunity.

The cell cycle-regulatory CDC25A phosphatase inhibits apoptosis signal-regulating kinase 1.

ABSTRACT CDC25A phosphatase promotes cell cycle progression by activating G1 cyclin-dependent kinases and has been postulated to be an oncogene because of its ability to cooperate with RAS to transform rodent fibroblasts. In this study, we have identified apoptosis signal-regulating kinase 1 (ASK1) as a CDC25A-interacting protein by yeast two-hybrid screening. ASK1 activates the p38 mitogen-activated protein kinase (MAPK) and c-Jun NH2-terminal protein kinase–stress-activated protein kinase (JNK/SAPK) pathways upon various cellular stresses. Coimmunoprecipitation studies demonstrated that CDC25A physically associates with ASK1 in mammalian cells, and immunocytochemistry with confocal laser-scanning microscopy showed that these two proteins colocalize in the cytoplasm. The carboxyl terminus of CDC25A binds to a domain of ASK1 adjacent to its kinase domain and inhibits the kinase activity of ASK1, independent of and without effect on the phosphatase activity of CDC25A. This inhibitory action of CDC25A on ASK1 activity involves diminished homo-oligomerization of ASK1. Increased cellular expression of wild-type or phosphatase-inactive CDC25A from inducible transgenes suppresses oxidant-dependent activation of ASK1, p38, and JNK1 and reduces specific sensitivity to cell death triggered by oxidative stress, but not other apoptotic stimuli. Thus, increased expression of CDC25A, frequently observed in human cancers, could contribute to reduced cellular responsiveness to oxidative stress under mitogenic or oncogenic conditions, while it promotes cell cycle progression. These observations propose a mechanism of oncogenic transformation by the dual function of CDC25A on cell cycle progression and stress responses.

Specific recognition of apoptotic cells reveals a ubiquitous and unconventional innate immunity

The purpose of physiological cell death is the noninflammatory clearance of cells that have become inappropriate or nonfunctional. Consistent with this function, the recognition of apoptotic cells by professional phagocytes, including macrophages and dendritic cells, triggers a set of potent anti-inflammatory responses manifest on multiple levels. The immediate-early inhibition of proinflammatory cytokine gene transcription in the phagocyte is a proximate consequence of recognition of the apoptotic corpse, independent of subsequent engulfment and soluble factor involvement. Here, we show that recognition is linked to a characteristic signature of responses, including MAPK signaling events and the ablation of proinflammatory transcription and cytokine secretion. Specific recognition and response occurs without regard to the origin (species, tissue type, or suicidal stimulus) of the apoptotic cell and does not involve Toll-like receptor signaling. These features mark this as an innate immunity fundamentally distinct from the discrimination of “self” versus “other” considered to be the hallmark of conventional immunity. This profound unconventional innate immune discrimination of effete from live cells is as ubiquitous as apoptotic cell death itself, manifest by professional and nonprofessional phagocytes and nonphagocytic cell types alike. Innate apoptotic immunity provides an intrinsic anti-inflammatory circuit that attenuates proinflammatory responses dynamically and may act systemically as a powerful physiological regulator of immunity.

Commitment and Effector Phases of the Physiological Cell Death Pathway Elucidated with Respect to Bcl-2, Caspase, and Cyclin-Dependent Kinase Activities

ABSTRACT Physiological cell deaths occur ubiquitously throughout biology and have common attributes, including apoptotic morphology with mitosis-like chromatin condensation and prelytic genome digestion. The fundamental question is whether a common mechanism of dying underlies these common hallmarks of death. Here we describe evidence of such a conserved mechanism in different cells induced by distinct stimuli to undergo physiological cell death. Our genetic and quantitative biochemical analyses of T- and B-cell deaths reveal a conserved pattern of requisite components. We have dissected the role of cysteine proteases (caspases) in cell death to reflect two obligate classes of cytoplasmic activities functioning in an amplifying cascade, with upstream interleukin-1β-converting enzyme-like proteases activating downstream caspase 3-like caspases. Bcl-2 spares cells from death by punctuating this cascade, preventing the activation of downstream caspases while leaving upstream activity undisturbed. This observation permits an operational definition of the stages of the cell death process. Upstream steps, which are necessary but not themselves lethal, are modulators of the death process. Downstream steps are effectors of, and not dissociable from, actual death; the irreversible commitment to cell death reflects the initiation of this downstream phase. In addition to caspase 3-like proteases, the effector phase of death involves the activation in the nucleus of cell cycle kinases of the cyclin-dependent kinase (Cdk) family. Nuclear recruitment and activation of Cdk components is dependent on the caspase cascade, suggesting that catastrophic Cdk activity may be the actual effector of cell death. The conservation of the cell death mechanism is not reflected in the molecular identity of its individual components, however. For example, we have detected different cyclin-Cdk pairs in different instances of cell death. The ordered course of events that we have observed in distinct cases reflects essential thematic elements of a conserved sequence of modulatory and effector activities comprising a common pathway of physiological cell death.

The Presumptive Phosphatidylserine Receptor Is Dispensable for Innate Anti-inflammatory Recognition and Clearance of Apoptotic Cells

The role of the presumptive phosphatidylserine receptor (PSR) in the recognition and engulfment of apoptotic cells, and the antiinflammatory response they exert, has been of great interest. Genetic deficiency of PSR in the mouse is lethal perinatally, and results to date have been ambiguous with regard to the phagocytic and inflammatory phenotypes associated with that deficiency. Recently, we found that the specific functional recognition of apoptotic cells is a ubiquitous property of virtually all cell types, including mouse embryo fibroblasts, and reflects an innate immunity that discriminates live from effete cells. Taking advantage of this property of fibroblasts, we generated, PSR+/+, PSR+/-, and PSR-/- fibroblast cell lines to examine definitively the involvement of PSR in apoptotic recognition and inflammatory modulation. Our data demonstrate that PSR-deficient cells are fully competent to recognize, engulf, and respond to apoptotic cells. Signal transduction in the responder cells, including the activation of Akt and Rac1, is unimpaired in the absence of PSR. We confirm as well that PSR is localized predominantly to the nucleus. However, it does not play a role in pro-inflammatory transcription or in the anti-inflammatory modulation of that transcriptional response triggered by apoptotic cells. We conclude that PSR is not involved generally in either specific innate recognition or engulfment of apoptotic cells.

Inhibiting Myosin Light Chain Kinase Induces Apoptosis In Vitro and In Vivo

ABSTRACT Previous short-term studies have correlated an increase in the phosphorylation of the 20-kDa light chain of myosin II (MLC20) with blebbing in apoptotic cells. We have found that this increase in MLC20 phosphorylation is rapidly followed by MLC20 dephosphorylation when cells are stimulated with various apoptotic agents. MLC20 dephosphorylation is not a consequence of apoptosis because MLC20 dephosphorylation precedes caspase activation when cells are stimulated with a proapoptotic agent or when myosin light chain kinase (MLCK) is inhibited pharmacologically or by microinjecting an inhibitory antibody to MLCK. Moreover, blocking caspase activation increased cell survival when MLCK is inhibited or when cells are treated with tumor necrosis factor alpha. Depolymerizing actin filaments or detaching cells, processes that destabilize the cytoskeleton, or inhibiting myosin ATPase activity also resulted in MLC20 dephosphorylation and cell death. In vivo experiments showed that inhibiting MLCK increased the number of apoptotic cells and retarded the growth of mammary cancer cells in mice. Thus, MLC20 dephosphorylation occurs during physiological cell death and prolonged MLC20 dephosphorylation can trigger apoptosis.

Involvement of Adenosine A2A Receptors in Engulfment-Dependent Apoptotic Cell Suppression of Inflammation

Efficient execution of apoptotic cell death followed by efficient clearance mediated by professional macrophages is a key mechanism in maintaining tissue homeostasis. Removal of apoptotic cells usually involves three central elements: 1) attraction of phagocytes via soluble “find me” signals, 2) recognition and phagocytosis via cell surface-presenting “eat me” signals, and 3) suppression or initiation of inflammatory responses depending on additional innate immune stimuli. Suppression of inflammation involves both direct inhibition of proinflammatory cytokine production and release of anti-inflammatory factors, which all contribute to the resolution of inflammation. In the current study, using wild-type and adenosine A2A receptor (A2AR) null mice, we investigated whether A2ARs, known to mediate anti-inflammatory signals in macrophages, participate in the apoptotic cell-mediated immunosuppression. We found that macrophages engulfing apoptotic cells release adenosine in sufficient amount to trigger A2ARs, and simultaneously increase the expression of A2ARs, as a result of possible activation of liver X receptor and peroxisome proliferators activated receptor δ. In macrophages engulfing apoptotic cells, stimulation of A2ARs suppresses the NO-dependent formation of neutrophil migration factors, such as macrophage inflammatory protein-2, using the adenylate cyclase/protein kinase A pathway. As a result, loss of A2ARs results in elevated chemoattractant secretion. This was evident as pronounced neutrophil migration upon exposure of macrophages to apoptotic cells in an in vivo peritonitis model. Altogether, our data indicate that adenosine is one of the soluble mediators released by macrophages that mediate engulfment-dependent apoptotic cell suppression of inflammation.

Physiological T-cell death: susceptibility is modulated by activation, aging, and transformation, but the mechanism is constant

It is not surprising that the recent explosion of interest in physiological cell death has been centered particularly on lymphocytes. Physiological cell death responses are singularly important in the biology of T lymphocytes, especially in the establishment and maintenance of a diverse, non-autoreactive, and self-limiting repertoire. Cell death responses can be triggered in T cells by a variety of stimuli; sensitivity to these inducers is altered as a function of differentiation, activation, aging, and transformation. The elimination of autoreactive T cells occurs by a process that involves comitogenic stimulation at high dose with antigenic and/or mitogenic agents. The control of susceptibility to this activation-driven cell death with differentiation and with prior activation provides a mechanistic explanation for the development of central and peripheral tolerance. Enhanced lymphocyte activation with aging also leads to an augmented activation-driven cell death response. However, aging does not alter cell death responses generally, and aging-associated changes in cell death responses cannot account for aging-associated immunopathology. Oncogenic transformation also alters the activation-driven cell death response by supplanting one of the required signals for activation-driven cell death. This difference provides a rationale for selective anti-tumor therapy. A single mechanism underlies all cases of physiological cell death and involves out-of-phase mitotic activity. We now know that of the two hallmarks of cell death, genome digestion is dispensable and mitotic-like events associated with cell cycle arrest are critical. T cells triggered to undergo physiological cell death arrest in a post-mitotic compartment of the cell cycle and die when they attempt a precocious and abortive mitosis.