Vimal A. Patel

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.

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.

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.

Rapamycin delays but does not prevent recovery from acute renal failure: role of acquired tubular resistance.

Background. We reported that rapamycin impairs recovery after acute renal failure (ARF) in rats. The objective of this study was to determine if recovery will eventually occur after ARF despite continued rapamycin treatment. Methods. ARF was induced in rats by renal artery occlusion. Glomerular filtration rate (GFR), morphology, and tubular cell proliferation were assessed either 2, 4, 6, or 7 days later. Rats were treated daily with rapamycin or vehicle throughout the study. Cultured mouse proximal tubular (MPT) cells were used to compare the antiproliferative effects of rapamycin after exposure for 1 and 7 days. Results. Two days after ARF, GFR was reduced severely but comparably in vehicle and rapamycin rats. In controls, GFR began to increase after day 2 and was normal by day 6. In rapamycin rats, GFR did begin to improve until after day 4 and reached normal values by day 7. In controls, many proliferating tubular cells were present in outer medulla on day 2, after which proliferation progressively decreased. By contrast, in rapamycin rats, proliferating cells were sparse on day 2, but then increased substantially through days 4 and 6. Cultured MPT cells exposed to rapamycin for 7 days were ∼10-fold more resistant to the antiproliferative effects of rapamycin than cells exposed for 1 day. Conclusions. Rapamycin delays but does not prevent renal recovery after ARF. MPT cells become resistant to rapamycin after prolonged exposure. We speculate that the ultimate recovery of renal function after ARF is due to the development of acquired tubular cell resistance to rapamycin.

Protein phosphatase 2A negatively regulates integrin alpha(IIb)beta(3) signaling.

Integrin αIIbβ3 activation is critical for platelet physiology and is controlled by signal transduction through kinases and phosphatases. Compared with kinases, a role for phosphatases in platelet integrin αIIbβ3 signaling is less understood. We report that the catalytic subunit of protein phosphatase 2A (PP2Ac) associates constitutively with the integrin αIIbβ3 in resting platelets and in human embryonal kidney 293 cells expressing αIIbβ3. The membrane proximal KVGFFKR sequence within the cytoplasmic domain of integrin αIIb is sufficient to support a direct interaction with PP2Ac. Fibrinogen binding to αIIbβ3 during platelet adhesion decreased integrin-associated PP2A activity and increased the phosphorylation of a PP2A substrate, vasodilator associated phosphoprotein. Overexpression of PP2Acα in 293 cells decreased αIIbβ3-mediated adhesion to immobilized fibrinogen. Conversely, small interference RNA mediated knockdown of endogenous PP2Acα expression in 293 cells, enhanced extracellular signal-regulated kinase (ERK1/2) and p38 activation, and accelerated αIIbβ3 adhesion to fibrinogen and von Willebrand factor. Inhibition of ERK1/2, but not p38 activation, abolished the increased adhesiveness of PP2Ac α-depleted 293 cells to fibrinogen. Furthermore, knockdown of PP2Acα expression in bone marrow-derived murine megakaryocytes increased soluble fibrinogen binding induced by protease-activated receptor 4-activating peptide. These studies demonstrate that PP2Ac α can negatively regulate integrin αIIbβ3 signaling by suppressing the ERK1/2 signaling pathway.

The affirmative response of the innate immune system to apoptotic cells

Growing evidence exists for a new role for apoptotic cell recognition and clearance in immune homeostasis. Apoptotic cells at all stages, irrespective of membrane integrity, elicit a signature set of signaling events in responding phagocytes, both professional and non-professional. These signaling events are initiated by receptor-mediated recognition of apoptotic determinants, independently of species, cell type, or apoptotic stimulus. We propose that the ability of phagocytes to respond to apoptotic targets with a characteristic set of signaling events comprises a second distinct dimension of innate immunity, as opposed to the traditional innate discrimination of self vs. non-self. We further propose that a loss or abnormality of the signaling events elicited by apoptotic cells, as distinct from the actual clearance of those cells, may predispose to autoimmunity.

AMPK protects proximal tubular cells from stress-induced apoptosis by an ATP-independent mechanism: potential role of Akt activation.

We examined the role of AMP-activated protein kinase (AMPK) in modulating the viability of cultured kidney proximal tubular cells subjected to metabolic stress induced by either dextrose deprivation, inhibition of glycolysis, or inhibition of mitochondrial respiration. We used BU.MPT cells, a conditionally immortalized kidney epithelial cell line derived from the proximal tubules of transgenic mice bearing a temperature-sensitive mutation of the simian virus 40 large-tumor antigen. All three forms of metabolic stress increased the phosphorylation and activity of AMPK. Activation of AMPK led to changes in the phosphorylation of two downstream targets of AMPK, acetyl coenzyme A carboxylase and p70 S6 kinase. Inhibition of AMPK, either pharmacologically with compound C (CC) or by gene silencing, significantly increased the amount of apoptosis in response to all three forms of metabolic stress. Although the amount of apoptosis was directly related to the severity of ATP depletion, inhibition of AMPK had no effect on cellular ATP levels. Notably, metabolic stress increased the phosphorylation and activity of Akt. Furthermore, inhibition of AMPK, with CC or gene silencing, abrogated the ability of metabolic stress to activate Akt. The augmentation of apoptosis induced by inhibition of AMPK was comparable to that induced by inhibition of Akt. We conclude that activation of AMPK following acute metabolic stress plays a major role in promoting the viability of cultured proximal tubular cells. Protection by AMPK appears to be due not to AMPK-mediated conservation of cell energy stores, but rather, at least in part, to AMPK-mediated activation of Akt.

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.

Apoptotic and necrotic cells as sentinels of local tissue stress and inflammation: Response pathways initiated in nearby viable cells

Virtually all cells in the body have the capacity to recognize and respond to dead cells. Viable cells discriminate apo from nec targets via distinct cell surface receptors. Engagement of these receptors induces “recognition-dependent” signaling events in viable responding cells that differ for apo vs. nec targets. Although “engulfment-dependent” signaling events also contribute to the response by viable cells, these events do not differ for apo vs. nec targets. While many signaling events are conserved across diverse cell lineages, other signaling events, especially those involving Akt, demonstrate lineage-specific variation. Whereas apo targets activate Akt in MΦ, they inhibit Akt in kidney epithelial cells. Differences in the responses to dead targets by viable migratory cells, such as MΦ, and viable fixed cells, such as kidney epithelial cells, permit cell-specific adaptations to local environmental change or stress. We propose that dead cells (apo and nec) act as sentinels to alert nearby viable cells to local environmental change or stress.

Abnormal regulation of the cytoskeletal regulator Rho typifies macrophages of the major murine models of spontaneous autoimmunity

Macrophages (mφ) from prediseased mice of all the major murine models of spontaneous autoimmunity have an identical defect in cytokine expression that is triggered by serum and/or apoptotic cells. We show here that mφ from prediseased mice of the same models of spontaneous autoimmunity share a serum‐dependent defect in the activity of Rho, a cytoplasmic G protein and cytoskeletal regulator. Affected strains include those developing lupus (BXSB, LG, MRL/l+, MRL/lpr, NZBWF1) and autoimmune diabetes (nonobese diabetic). No similar defect in Rho activity occurred in seven control strains. In the presence of serum, Rho activity in mφ from all autoimmune‐prone strains was reduced to less than 10% of that in control mice. In contrast, under serum‐free conditions, Rho activity was ompletely normal in autoimmune‐prone mφ. The activities of Ras, another cytoplasmic G protein, and Rac and Cdc42, two additional G protein regulators of the cytoskeleton, were regulated normally in autoimmune‐prone strains. Serum‐dependent dysregulation of Rho was associated with multiple abnormalities, including increased adhesion to various surfaces, a more spread dendritic morphology, and an altered actin cytoskeletal organization. Our results suggest that mφ from multiple, genetically diverse, autoimmune‐prone strains share a mutation or allelic difference affecting signal transduction within a specific Rho‐regulatory pathway.