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Department of Microbiology and Immunology, University of Illinois College of Medicine, Chicago, IL 60612
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cells that die in these physiological contexts typically are removed rapidly by phagocytic cells, including macrophages (1, 8, 9). The phenomenon of dead cell clearance was recognized contemporaneously with the earliest observations of cell death in the 1880s (reviewed by Clarke and Clarke in Ref. 10). Metchnikoff (11) was the first to describe the phagocytic process, performed by what he termed microphages (neutrophils) and macrophages. From that time, the process of phagocytosis has been associated typically with inflammatory responses to pathogens. The most significant aspect of the process of physiological cell death, in contrast, is its targeted elimination of cells without inflammation or pathology (12, 13).
The first demonstration of the absence of an inflammatory effect of apoptotic cell phagocytosis came from an examination of the proinflammatory mediators released from macrophages following their ingestion of dying neutrophils in culture (14). Neutrophils are themselves short-lived phagocytic polymorphonuclear granulocytes (Metchnikoff’s microphages); morphological studies had suggested previously that the granular contents of neutrophils were not released when they died (9), leading to the view that the avoidance of cellular leakage, especially by phagocytic ingestion before lysis, is an important aspect of the physiological cell death process and a key to the regulation of inflammation. Whereas opsonized erythrocytes were engulfed and promoted the secretion of two early markers of the macrophage inflammatory response (an arachidonic acid-derived inflammatory mediator and a granule enzyme), apoptotic neutrophils did not trigger release, although they were engulfed efficiently.
Subsequent work demonstrated that a number of cytokines and chemokines associated with inflammation (IL-6, IL-8, monocyte chemoattractant protein (MCP)3-1, TNF-
, and GM-CSF) were not secreted from phagocytes that engulfed apoptotic targets (15, 16, 17). Those studies also demonstrated that the lack of inflammatory responses associated with engulfment of apoptotic corpses is more than a passive avoidance of stimulation: apoptotic cells affirmatively inhibit inflammatory responses from the phagocytic cells that ingest them. Whereas stimulation of phagocytic cells via the Toll-like receptor (TLR)4 signaling complex (18) upon engagement with bacterial LPS triggers significant cytokine secretion (especially including TNF-, IL-1
, and IL-6), the additional ingestion of apoptotic cells attenuates this response (16, 17, 19). This inhibitory property appears to be a common attribute acquired posttranslationally by cells dying physiologically, regardless of the cell type or the particular death stimulus (17).
We have shown that the inhibitory effect of apoptotic targets is dominant in trans to the stimulatory effect of necrotic cells and/or LPS (17), indicating unambiguously that this property represents an apoptotic gain-of-function. This dispels the notion that the phagocytosis of apoptotic cells must occur prelytically so as to circumvent the inflammatory response that ensues following the release of noxious intracellular contents from ruptured corpses (20, 21, 22). Cells that die physiologically acquire an anti-inflammatory activity that persists even as the cells lose membrane integrity (17).
Thus far, the underlying mechanistic aspects of this process remain unresolved. It is not clear whether modulation is exerted principally on a transcriptional or posttranscriptional level, or what the primary target of regulation might be. Results from previous studies exploring the regulation of cytokine and chemokine gene transcripts by apoptotic targets have not been definitive. For example, Fadok et al. (16) reported that the abundance of TNF- transcripts in LPS-stimulated macrophages, assessed by RT-PCR, was diminished >75% following phagocytosis of apoptotic targets, whereas McDonald et al. (23), using an RNase protection assay, reported no change in the level of TNF- mRNA following target phagocytosis. In part, these ambiguities likely are due to complications associated with the analysis of mixed populations of responder and target cells.
In this report, we describe results from new studies in which we have addressed these issues by examining transcriptional events within macrophages specifically. Our results demonstrate that apoptotic modulation involves the regulation of macrophage transcription and represents direct transcriptional control through an innate signaling pathway triggered upon the recognition of apoptotic determinants.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Murine macrophage cell line RAW 264.7 and DO11.10 murine T hybridoma cells were grown at 37°C in a humidified, 5% (v/v) CO2 atmosphere in RPMI 1640 medium (Mediatech, Herndon, VA) supplemented with heat-inactivated FBS (10% v/v; HyClone Laboratories, Logan, UT), 2 mM L-glutamine, and 50 µM 2-ME. Human epithelial HeLa cells were grown in DMEM with 4.5 g/L glucose (Mediatech) supplemented with FBS (10% v/v), 2 mM L-glutamine, and 50 µM 2-ME, and Chinese hamster ovary (CHO) cells were grown in -MEM (Life Technologies, Grand Island, NY) supplemented only with FBS (10% v/v).
Physiological cell death (apoptosis) was induced by treatment of target cells with the macromolecular synthesis inhibitors actinomycin D (200 ng/ml, 12 h) or cycloheximide (1 µg/ml, 12 h; see Ref. 24). Cells were killed pathologically (necrotic death) by incubation at 55°C for 20–25 min (until trypan blue uptake indicated compromise of membrane integrity) (17). In all cases, target cells (viable, apoptotic, and necrotic) were washed in complete medium before addition to macrophages.
The single-cell cytofluorimetric analyses of Asp-Glu-Val-Asp (DEVD)-specific caspase activity (representing the activity of caspases 3 and 7), using the intracellular caspase substrate PhiPhiLux-G2D2 (OncoImmunin, Gaithersburg, MD), and mitochondrial membrane potential, monitored with tetramethylrhodamine ethyl ester, during the process of cell death have been previously described (24, 25). In some cases, target cells were fixed by treatment with formaldehyde before their interaction with macrophages. Target cells that had been washed twice in PBS were incubated in formaldehyde (125 mM in PBS; Polysciences, Warrington, PA) for 20 min at 25°C. Fixed cells were again washed twice in complete medium before addition to macrophages.
Phagocytosis assay
Target cells were labeled green with CFSE (0.2 µM; Molecular Probes, Eugene, OR) and were then induced to undergo cell death or were left untreated. Macrophages were labeled red with 5-(and 6-)-(((4-chloromethyl)benzoyl)amino)tetramethylrhodamine (CMTMR, 10 µM; Molecular Probes). Labeled macrophages were cocultured with target cells at the indicated macrophage to target ratio for 1 h at 37°C. Cells were harvested with PBS supplemented with 0.4 mM Na2EDTA and analyzed cytofluorimetrically on a FACSCaliber instrument (BD Biosciences, San Jose, CA). Cells with macrophage-like scatter properties that were both CMTMR-positive (Ex
= 488 nm, Em = 610 nm ± 15 nm) and CFSE-positive (Ex = 488 nm; Em = 530 ± 15 nm) represented macrophages that had engulfed targets. Engulfment is calculated as the fraction of double-positive macrophages (all CMTMR-positive cells that also are CFSE-positive). Results are expressed as the formula: specific engulfment = (fraction of engulfing macrophages with apoptotic targets) - (fraction of engulfing macrophages with viable targets). Note that these cytofluorimetric procedures only monitor engulfment of target cells. Targets that are bound but not engulfed are disrupted and do not remain adherent during the analysis, although they could be enumerated under static microscopic examination (17).
Real-time RT-PCR analysis
Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA). To insure that RNA was derived from equivalent samples, each cell mixture contained fixed numbers of macrophages and target cells (the target to macrophage ratio was 10:1), and no cells were removed before lysis. For determinations of transcript levels in macrophages alone, target cells were added only after incubation and immediately before lysis. Each sample was analyzed independently in quadruplicate wells. cDNA synthesis (starting with 0.1 µg of RNA for the analysis of TNF- and 1.0 µg of RNA for the analysis of IL-6) and PCR were performed in the same well using Taqman Gold RT-PCR reagents and Taqman (FAM-490) probes and primers (Applied Biosystems, Foster City, CA). The iCycler system (BioRad, Hercules, CA) was used for amplification and data collection. Amplifications for each RT-PCR primer/probe set were calibrated with the relevant cytokine cDNA clone (26, 27) over five orders of magnitude by linear regression according to the formula: CN = 10-[(TC - b)/m], where CN is the template copy number and TC is the observed threshold crossing point. The standard curves are shown in Fig. 1. The derived constants for IL-6 are m = -3.718 and b = 42.587 and for TNF- are m = -2.886 and b = 37.151.
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Transfections and luciferase assays
Macrophages were plated at 1.4 x 106 cells per 100-mm diameter dish. The next day, cells were cotransfected with 3 µg of pNF
B-Luc, a plasmid containing the firefly Photinus pyralis luciferase gene the expression of which is driven by a basal transcriptional promoter linked to four copies of the B motif (Clontech Laboratories, Palo Alto, CA), together with 0.1 µg of pRL-SV40, a Renilla reniformis luciferase control vector the constant expression of which is dependent on the SV40 early enhancer/promoter region (Promega, Madison, WI), using Effectene Transfection Reagent (Qiagen, Valencia, CA). Similar transfections were performed with pAP1-Luc (Clontech), a plasmid with which firefly luciferase expression is dependent on the four tandem copies of the AP-1 enhancer region. In some experiments, plasmid DNAs encoding p300 or CREB binding protein (CBP) also were included in the cotransfections. Titrated constructs, together with empty vector, totaled an additional 1.6 µg of DNA per 100-mm dish.
After 16 h, cells were replated in 6-well plates (2.0 x 105 cells/well). Following an additional 8 h, macrophages were incubated without or with the indicated target cells (at a target cell to macrophage ratio of 10:1) and/or LPS (1 µg/ml, Escherichia coli O111:B4; Sigma-Aldrich, St. Louis, MO). In some experiments, recombinant human TGF1 (10 ng/ml; R&D Systems, Minneapolis, MN) and/or pan-TGF-specific blocking Abs (AB-100-NA, 100 µg/ml; R&D Systems) were included. Cell extracts were prepared after further incubation (5 h or as indicated), and luciferase activities were measured by the Dual Luciferase Reporter Assay System (Promega) in an FB12 Luminometer (Zylux, Oak Ridge, TN). Each condition was repeated in triplicate wells, and the luciferase activities in cells from each well were determined independently. Within one experiment, Renilla luciferase activities among samples varied <8%. The firefly luciferase activity in each sample was normalized relative to the internal Renilla luciferase activity, and the relative level of normalized firefly luciferase activity (compared with the activity in an untreated population) was taken as a measure of the activity of the relevant (NF-B or AP-1) transcriptional activator.
Cellular extract preparation and immunoblot analysis
Total cellular extracts were prepared in RIPA buffer (1x PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) with protease inhibitors (Complete Protease Inhibitor Cocktail; Roche Diagnostics, Indianapolis, IN). For preparation of nuclear extracts, cells were incubated for 4 min on ice in buffer I (10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM NaCl, 0.4% Nonidet P-40, and Complete Protease Inhibitor Cocktail) and centrifuged (5 min at 4°C, 600 x g). Nuclei were resuspended in buffer II (20 mM HEPES pH 7.9, 20% glycerol, 0.4 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA) and disrupted for 30 min at 4°C. Supernatants were collected after centrifugation (10 min at 4°C, 13,000 x g). A total of 20 µg of each protein sample (determined by the Bradford method; BioRad) were run on 12% SDS polyacrylamide gels and transferred to HyBond C-Extra (Amersham, Arlington Heights, IL). Blots were blocked with 5% dry milk in PBS before probing with IB-specific (SC-371, 1/300 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) or NF-B p65 (RelA) subunit-specific (SA-171, 1/100 dilution; BioMol, Plymouth Meeting, PA) Abs. Following incubation with appropriate secondary Abs conjugated to HRP (1/1000 dilution, Santa Cruz Biotechnology), immunoreactive bands were visualized by the luminol reaction (ECLplus; Amersham).
Cognate DNA-binding assay
Cognate DNA binding of NF-B was assessed using an ELISA-based assay (Trans-Am NF-B p65; Active Motif, Carlsbad, CA). Whole cell extracts (20 µg of protein/20-µl reaction) were incubated with a double-stranded oligonucleotide containing the NF-B consensus site (5'-GGGACTTTCC-3') that had been immobilized in wells of a 96-well plate. In some experiments, excess soluble oligonucleotide (20 pMol per reaction) was included as a competitor. After washing, bound NF-B was detected with a p65 RelA-specific Ab. The reaction was developed colorimetrically with an HRP-conjugated secondary Ab and quantified spectrophotometrically at 450 nm (Microplate Autoreader Model EL311; Bio-Tek Instruments, Winooski, VT).
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Results |
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Our previous results suggested that the blockade of proinflammatory cytokine release from macrophages by apoptotic cells was virtually immediate, and that this apoptotic cell control might be exerted upstream or independent of proinflammatory TLR signaling (17). To test this hypothesis and examine on what level apoptotic modulation is enforced, we have used real-time RT-PCR analysis to quantify specific cytokine and chemokine gene transcript levels in macrophages unambiguously. We have noted that the uncertain findings from transcriptional analyses reported previously (16, 23) likely are due to complications associated with the analysis of mixed populations of responder and target cells. In an effort to overcome the technical obstacles posed by cellular heterogeneity and insure that RNA was derived from equivalent samples, we used clonal cell lines of macrophages and targets, and each cell mixture contained a fixed numbers of macrophages and target cells (the macrophage to target cell ratio in these experiments was 1:10). For determinations of transcript levels in macrophages alone, target cells were added immediately before lysis.
Using this strategy, we were able to quantify cytokine gene transcripts per macrophage in absolute terms (see Materials and Methods). As shown in Fig. 2, A and B, our results indicate that, on average, a RAW 264.7 macrophage cell stimulated with LPS for 2 h harbors 
5300 TNF- transcripts and one IL-6 transcript. On average, each uninduced macrophage cell expresses 220 TNF- transcripts and 0.06 IL-6 transcripts. Thus, stimulation of macrophages via TLR4 with bacterial LPS results in the rapid up-regulation of proinflammatory cytokine gene transcripts, in the cases of IL-6 and TNF-, 20-fold induction within 2 h, consistent with results of Hoshino et al. (18). (The DO11.10 targets harbor 7 TNF- transcripts and <0.003 IL-6 transcripts per cell). The very low abundance of IL-6 transcripts, which accords with previous results (28, 29), suggests that the response may be limited to only some macrophages within the population, what we have referred to as "jackpots" (17).
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transcripts is reduced by at least 45% within 2 h when macrophages encounter apoptotic targets (Fig. 2, A and B). Thus, the modulation by apoptotic corpses of TNF- and IL-6 secretion that we observed previously (17) faithfully recapitulates their more rapid regulation of TNF- and IL-6 transcripts in macrophages.
We also have observed that the phagocytosis of apoptotic targets results in the diminution of macrophage inflammatory protein-1 transcripts within LPS-stimulated macrophages, but not in an alteration in the abundance of transcripts of the chemokine MCP-1 (data not shown). These patterns of transcript regulation, too, are reflective of the observed effects of phagocytosis on the secretion of those factors (23, 30, 31). Together, these results raise the possibility that the modulation of inflammatory responses by apoptotic cells may be effected primarily on the level of specific gene transcription.
The modulation of inflammatory cytokine gene transcript levels is an immediate-early response of macrophages to apoptotic corpses
We asked whether the modulation of cytokine gene transcripts within the responding macrophage is a primary response to the apoptotic target, one that can occur independently of new protein synthesis. Macrophages were pretreated with cycloheximide, an inhibitor of translation, for 90 min before the addition of apoptotic targets and LPS, and cycloheximide treatment was continued for the duration of their interactions. The data in Fig. 2, C and D, reveal that both the LPS-induced activation and the down-regulation by apoptotic corpses of TNF- and IL-6 gene transcripts are immediate-early responses of the macrophage, occurring in the absence of translation. Although idiosyncratic effects of translational blockade on levels of cytokine gene transcripts were evident from the real-time RT-PCR analyses (the induction of TNF- mRNA by LPS was enhanced, whereas the abundance of IL-6 transcripts in all cases was reduced about 10-fold), the relative extent of apoptotic modulation was unaltered.
The observation that the induction of cytokine gene expression ensues in the absence of protein synthesis is consistent with previous studies (32, 33, 34). In particular, transcriptional activators have been shown to be the direct targets of signal transduction pathways initiated by TLR4 and other Toll and IL-1R domain-containing receptors that involve myeloid differentiation factor 88-like adapter molecules and members of the IL-1R-associated signal kinase family (34, 35, 36, 37, 38). The finding that the modulatory effect exerted by apoptotic cells similarly occurs without need of new protein synthesis suggests that apoptotic cells exert their anti-inflammatory effect on the level of transcriptional regulation primarily, and that a comparably direct signal transduction pathway, leading from apoptotic recognition to transcriptional modulation, exists within the macrophage.
A reporter of NF-B-dependent transcription reliably reveals the modulatory effect exerted by apoptotic targets
Because the regulation of cytokine gene transcription signaled through TLR4 and other inflammatory receptors is effected by critical transcriptional activators such as NF-B (39, 40), we asked whether the modulation of inflammatory responses exerted by apoptotic cells might target NF-B itself. We transiently transfected macrophages with pNFB-Luc, a plasmid containing the firefly luciferase gene the expression of which is driven by a basal transcriptional promoter linked to four copies of the B motif. Macrophages were cotransfected with a constitutive (NF-B-independent) Renilla luciferase control vector, which served as an internal normalization control. Following transfection, macrophages were incubated with target cells and LPS, and firefly and Renilla luciferase activities then were measured.
As indicated by these luciferase reporters, LPS-activated NF-B-dependent transcription in macrophages is inhibited specifically following their interaction with apoptotic cells (Fig. 3A). Apoptotic target interactions do not lead to a global repression of macrophage transcription, however; note that expression of the cotransfected Renilla luciferase control vector, like MCP-1 mRNA, is unaffected by apoptotic targets (see Materials and Methods). Viable and necrotic cells do not exert such inhibition; indeed, necrotic cells slightly enhance macrophage NF-B activity, just as they stimulate LPS-dependent cytokine release (17). In contrast to cell lysates (41, 42), authentic necrotic cells alone are insufficient to trigger such responses.
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B-dependent macrophage transcription by apoptotic cells is readily evident within 5 h of cellular interaction using this approach; the luciferase signal is insufficient at earlier times to be reliably informative. The extent to which apoptotic targets attenuate NF-B-dependent macrophage transcription becomes increasingly profound with longer times of cellular interaction (compare <60% inhibition at 5 h with >80% inhibition at 18 h; Fig. 3, A and B). That the extents of LPS stimulation and apoptotic attenuation of NF-B-dependent transcription vary independently with time suggests that independent signaling pathways are involved in these two responses.
It is striking that a single pattern of modulation of macrophage responsiveness by apoptotic cells is evident by three independent measures: cytokine secretion, gene transcript levels, and transcriptional activity (Figs. 2 and 3A; Ref. 17). This specific response is elicited by apoptotic cells generally, independent of the particular death-inducing stimulus (Fig. 3A, Ref. 17 , and data not shown). Moreover, the modulatory activity of apoptotic cells does not appear to manifest species-specific restriction. We and other investigators have shown that determinants for recognition were conserved between human and murine cells (17, 19, 23). The data in Fig. 3C demonstrate as well that this conservation among mammalian species is evident with respect to determinants for NF-B-dependent transcriptional modulation.
Modulation of macrophage responsiveness is a direct consequence of apoptotic cell recognition, independent of engulfment and soluble TGF
We have shown previously that the recognition of apoptotic and necrotic cells by macrophages involves distinct and noncompeting mechanisms, and that the discrimination between these two classes of targets occurs on the level of their binding to the engulfing cell (17). We wondered whether the modulation of inflammatory responsiveness would be signaled as a direct consequence of the specific recognition of apoptotic cells, independent of their engulfment. Because phagocytosis does not occur at 4°C, we were able to dissociate binding from engulfment simply as a function of incubation temperature in our earlier studies. However, transcription also is effectively blocked at low temperature. We therefore have taken a different approach, using cytochalasin D, a pharmacologic agent that inhibits actin polymerization, to abrogate engulfment specifically. Using fluorescence assays to assess binding and engulfment (17), we have confirmed that treatment of macrophages with cytochalasin D abrogates target cell engulfment without affecting specific binding (Fig. 4A and data not shown). Strikingly, the specific binding of apoptotic cells to macrophages without engulfment is entirely sufficient for the manifestation of their modulatory activity (Fig. 4B). These experiments further the view that apoptotic cells acquire cell-associated determinants for recognition and anti-inflammatory signaling during the process of physiological cell death, and that specificity is exerted on the level of recognition (17).
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(16, 23, 30). The data presented in Fig. 4C recapitulate the ability of TGF to mimic the modulatory effect of apoptotic cells on macrophages, as measured in this study with respect to LPS-activated NF-B-dependent transcription, and further attest to the efficacy of TGF-specific Abs in blocking TGF action (16, 31). Notably, the ability of apoptotic targets to modulate macrophage responsiveness is undiminished in the presence of those same TGF-specific blocking Abs (Fig. 4C). Together, these data emphasize that the modulation of inflammatory responsiveness in macrophages is initiated as a direct consequence of their specific recognition of apoptotic cells.
The inhibition of NF-B activation per se does not appear to be the molecular target of apoptotic modulation
In light of the observed diminution of NF-B-dependent transcriptional activity, we asked whether apoptotic interactions with macrophages interfere with the activation of NF-B itself. Degradation of IB, the major inhibitor of NF-B, is the proximal target of TLR4/myeloid differentiation factor 88/IL-1R-associated kinase signaling (43). We examined IB degradation specifically in macrophages by exploiting the electrophoretic distinction between human and murine IB and the ability of human targets to modulate murine macrophage responses (Fig. 3B). As shown in Fig. 5A, the rapid degradation of macrophage IB ensues normally in LPS-stimulated macrophages regardless of their interaction with apoptotic targets. (In this experiment, the human target cells, induced to die with actinomycin D, had not degraded their IB as they died). The interaction with apoptotic cells also causes no diminution of the LPS-stimulated recruitment of NF-B to the nucleus (data not shown) or the activation of cognate DNA sequence-specific DNA-binding activity (Fig. 5B), consistent with the DNA-binding data of McDonald et al. (23). These results suggest that apoptotic cells exert a down-regulation of NF-B-dependent transcription by a mechanism distinct from the inhibition of NF-B activation. Our results also indicate that the proximal steps of TLR-specific signaling are unimpaired by macrophage interactions with apoptotic targets.
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B- and AP-1-dependent transcription raises the possibility that a common transcriptional coactivator, such as CBP or its paralogue p300 (45), could be the target of apoptotic modulation. Results from preliminary experiments are consistent with such a model. In a dose-dependent manner, and at concentrations that do not trigger squelching (46), transfection of p300 or CBP relieves the inhibition of NF-B-dependent transcription in macrophages exerted normally by apoptotic corpses (Fig. 6B and data not shown).
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We have begun to characterize the nature of the putative apoptotic determinant(s) for recognition and anti-inflammatory signaling. Previous work had suggested that the fixation of apoptotic cells with paraformaldehyde (4%) did not interfere with their recognition by macrophages (47) nor did it cause the appearance of apoptotic-like modulatory activity on viable cells (19). We find that apoptotic modulatory activity similarly is stable to fixation with formaldehyde (Fig. 7A).
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2 h in the case of actinomycin D-induced death; Fig. 7B) likely reflects the adventitious appearance of a small number of cells most accelerated in the death response expressing potent modulatory activity. These results are consistent with observations that dying cells interact rapidly with phagocytes, leading to their noninflammatory clearance, and suggest that the acquisition of modulatory activity is linked to the activation of effector phase enzymes within the dying cell. |
Discussion |
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The conserved and cell autonomous (49, 50) process of physiological cell death assures the specifically targeted removal of dying cells by engulfing phagocytic cells in the absence of infiltrating immune effectors. The efficient noninflammatory clearance of inappropriate T cells during development in the thymus illustrates this phenomenon dramatically (1). In contrast, pathological (necrotic) cell death, marked by rapid, disorganized swelling and rupture and associated with pathological tissue injury (51), elicits inflammatory responses in addition to phagocytic clearance (21, 51).
Cells undergoing physiological cell death are recognized specifically by macrophages (and other phagocytes), and exert immediate-early down-regulation of proinflammatory transcription in those cells. Our demonstration that apoptotic modulation occurs in the absence of new translation in the macrophage strongly implies that this likely is the primary mode by which noninflammatory clearance of apoptotic corpses is initiated. Our data also suggest that this response occurs independently of proximal steps of TLR signaling. The ability of transfected transcriptional coactivators to relieve this modulation raises the intriguing possibility that the recognition of apoptotic cells triggers the sequestration of these or other requisite elements away from proinflammatory transcriptional complexes in stimulated macrophages.
It is striking that apoptotic cells, regardless of the particular suicidal stimulus that triggered the cell death, are recognized by macrophages and exert modulatory function without species-specific restriction. The discrimination of "self" from "stranger", leading to the elimination of the latter typically in a phlogistic context, is considered to be the major function of innate immunity. In this study we show that discrimination of live from effete cells, without regard to self, is a distinct innate immune function.
These results extend our conclusions that conserved modulatory determinants arise during the cell death process in the absence of macromolecular synthesis, and reveal that their effect is exerted in the absence of engulfment. Like the induction of death-associated effector activities (24), the appearance of determinants for recognition and inhibition of inflammation represents a posttranslational gain-of-function, the revelation of cryptic anti-inflammatory activity, during the process of physiological cell death (17). Informed by these results, we make the explicit hypothesis that cells undergoing physiological cell death express apoptotic determinant(s) for response modulation, and that phagocytic cells express a specific (signaling) receptor(s) for these apoptotic cell surface determinants. Further, in this model, the new expression of apoptotic modulatory activity, leading to cellular clearance within a physiological context, represents an apoptotic gain-of-function that is the definitive point of irreversible death commitment (48).
Anti-inflammatory modulation and soluble mediators
Several groups have proposed that soluble mediators serve as proximal intermediates that elicit the anti-inflammatory response in macrophages, akin to their role in endotoxin tolerance (52). For example, Voll et al. (19) observed that the release of IL-10 from LPS-stimulated macrophages coincided with the inhibition of secretion of TNF-, IL-1, and IL-12 following their interaction with apoptotic cells. Fadok et al. (16) reported a similarly inverse relationship between the stimulation of TGF, platelet-activating factor, and PGE2 release and the check of TNF-, IL-1, and several other inflammatory cytokines and lipids by macrophages following engulfment of apoptotic targets. IL-10 secretion was found not to be enhanced generally by apoptotic targets, however (16, 23). Provocatively, although the pharmacologic blockade of platelet-activating factor, and PGE2 signaling had only slight effect (23), the addition of TGF-specific neutralizing Ab to cultures of macrophages and apoptotic targets partially restored proinflammatory cytokine release (16, 23), leading to a model in which subsequent inflammatory down-regulation is dependent on initial TGF stimulation (16, 23, 30). It is important to note that macrophage responses were assessed in those studies after long periods (
18 h) of target cell interaction.
In contrast, our analyses at earlier times indicate that TGF is not involved in the initial modulation of inflammatory responses by apoptotic cells. Indeed, we have argued previously that an autocrine or paracrine mode for initiation of the anti-inflammatory response is not obviously consistent with the rapid kinetics of apoptotic modulation (17). Independent refutation of a primary role for TGF comes from measures of TGF-regulated transcriptional activity (our unpublished observations). Because TGF has been reported to synergize with LPS to enhance AP-1 activity (31), our observation that AP-1 activity is inhibited acutely in LPS-stimulated macrophages that interact with apoptotic cells also is consistent with the absence of TGF function at these early times. Our results suggest that the initiation of inflammatory modulation occurs as a direct consequence of the interaction of apoptotic target cells with macrophages, and without the involvement of TGF.
A secondary role for TGF, acting at later times to amplify and disseminate the modulatory response, is not excluded by these findings. Our data demonstrate that the acquisition of determinants for their specific recognition by macrophages and their ability to exert inflammatory modulation occur in dying cells early within the effector phase of the physiological cell death process, well before the plasma membrane is compromised (also see Ref. 17). The observation of Chen et al. (53) that preformed TGF may be released directly from dying lymphoid target cells late in the cell death process, at a time when cellular integrity is compromised, is consistent with this view and with evidence that the anti-inflammatory phenotype of apoptotic cells is sustained throughout the process of cellular dissolution (17). It may be that such a paracrine function serves to assure the modulation of inflammation even if the clearance of apoptotic cells is delayed.
Determinants for apoptotic recognition and modulation
Properties unique to the dying cell determine the mode and outcome of phagocytosis. We showed previously that discrimination between apoptotic and necrotic corpses occurs by distinct and noncompeting mechanisms on the level of binding, in a temperature-independent manner dissociated from engulfment (Ref. 17 , but see Ref. 54). Further, determinants of apoptotic target recognition are acquired posttranslationally during the process of physiological cell death (17, 24).
Although numerous putative apoptotic ligands, including carbohydrates, lipids, and oxidized proteins (8, 21, 55, 56, 57), and phagocyte receptors, including integrins, scavenger receptors such as CD36 and CD68, CD14, and tethering molecules such as thrombospondin and components of the complement system (21, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67) have been implicated (13), the functional analysis of these putative ligands and receptors, in many cases, has been incomplete. Most functional tests have involved measures of apoptotic target "uptake," without discrimination between actual recognition and engulfment. In those cases in which functional roles were evaluated, the actions of the identified molecules (for example, exposed phosphatidylserine, the class A scavenger receptor, the ATP binding cassette-type transporter ABC1, and the Mer tyrosine kinase) (17, 47, 61, 68, 69) were found to be limited to the phagocytic process and not to involve recognition.
Genetic studies of cell death in Caenorhabditis elegans are illuminating in this context. Engulfment in C. elegans has been shown to involve the products of at least seven genes. These gene products function in multiple and redundant pathways for the clearance of dead cells (70), and their orthologues have been implicated in the clearance of dead cells in mammals. For example, Ced1 is related to an endothelial cell scavenger receptor, Ced5 is an orthologue of DOCK180, involved in cytoskeletal rearrangement and process extension, and Ced7 is the orthologue of ABC1 (71, 72, 73). Recent work demonstrates that the pathways encoded by these genes in C. elegans are involved in the clearance of both apoptotic and necrotic corpses (74). In light of the finding that apoptotic and necrotic cells are recognized by distinct mechanisms (17), we argue that all of these genes must encode molecules involved in engulfment, and not in recognition. By extension, the mammalian counterparts of these genes similarly encode molecules for which functions must be limited to phagocytic processes distinct from recognition. The known functions of the gene products already identified are consistent with this argument.
To date then, the identities of the specificity determinants of apoptotic cells and the recognition elements of phagocytes remain elusive. If engulfment has been an insufficiently stringent criterion for the identification of those molecules, it may be that the modulation of inflammatory responses associated with apoptotic cell clearance holds greater promise. Indeed, its ability to be removed without inflammation is the uniquely defining property of the apoptotic corpse.
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Acknowledgments |
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cDNA clones, respectively, to Jim Cook (University of Illinois College of Medicine) for pNFB-Luc, pAP1-Luc, p300, and CBP plasmids, and to Akira Komoriya and Beverly Packard (OncoImmunin, Gaithersburg, MD) for the DEVD-specific caspase substrate PhiPhiLux-G2D2. We thank our colleagues Karen Colley, Peter Henson, Tom Hope, Amy Kenter, Tone Sandal, John Varga, William Walden, and Richard Ye for their constructive comments. |
Footnotes |
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2 Address correspondence and reprint requests to Dr. David S. Ucker, Department of Microbiology and Immunology, University of Illinois College of Medicine, Room E803 (Mail Code 790), 835 South Wolcott, Chicago, IL 60612. E-mail address: duck{at}uic.edu
3 Abbreviations used in this paper: MCP, monocyte chemoattractant protein; CBP, CREB binding protein; CMTMR, 5-(and 6-)-(((4-chloromethyl)benzoyl)amino)tetramethylrhodamine; TLR, Toll-like receptor.
Received for publication July 8, 2003. Accepted for publication November 3, 2003.
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) were performed as described in Materials and Methods. The relevant cloned cytokine cDNAs, over an input range of five orders of magnitude, were used as templates. Data were analyzed by linear regression, according to the formula CN = 10-[(TC-b)/m] (where CN is the template copy number, and TC is the observed threshold crossing point), to generate standard calibration curves.
) also was added to macrophages 90 min before other additions. Data are presented as macrophage-specific transcript levels relative to the uninduced control population ("fold-induction"). TNF-
) (A) or 18 h (mesh bar) (B), and luciferase activities were measured. Data are presented as normalized luciferase activities in treated macrophages relative to the uninduced control population ("fold-induction"). NF-
) 2 µM cytochalasin D for 45 min before the addition of apoptotic or viable targets (at a target to macrophage ratio of 20:1) and LPS (1 µg/ml), as indicated. Cell extracts were prepared after 5 h and luciferase activities were measured. C, NF-
) were incubated without or with apoptotic human HeLa cell targets (that had been induced to die by treatment with actinomycin D) for 1 h before the addition of LPS (1 µg/ml) as indicated. After an additional 10 min of incubation, cells were lysed. The presence of I
) and the loss of mitochondrial membrane potential (