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Originally published In Press as doi:10.1074/jbc.M605146200 on October 20, 2006

J. Biol. Chem., Vol. 281, Issue 50, 38376-38384, December 15, 2006
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Apoptotic Cells, through Transforming Growth Factor-beta, Coordinately Induce Anti-inflammatory and Suppress Pro-inflammatory Eicosanoid and NO Synthesis in Murine Macrophages*

Celio G. Freire-de-Lima{ddagger}§12, Yi Qun Xiao§1, Shyra J. Gardai§, Donna L. Bratton§, William P. Schiemann§, and Peter M. Henson§3

From the {ddagger}Instituto de Biofísica Carlos Chagas Filho, Federal University of Rio de Janeiro, Rio de Janeiro RJ 21944-970, Brazil and §Program in Cell Biology, Department of Pediatrics, National Jewish Medical and Research Center, Denver, Colorado 80206

Received for publication, May 30, 2006 , and in revised form, October 11, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Apoptotic cells are rapidly engulfed by adjacent tissue cells or macrophages before they can release pro-inflammatory/proimmunogenic intracellular contents. In addition, recognition of the apoptotic cells is actively anti-inflammatory and anti-immunogenic with generation of anti-inflammatory mediators such as transforming growth factor-beta (TGF-beta) and anti-inflammatory eicosanoids. Here, we have investigated the role played by the induction of TGF-beta in the coordinate expression of anti-inflammatory eicosanoids or peroxisome proliferator-activated receptor-{gamma} and in the suppression of pro-inflammatory lipid mediators and nitric oxide (NO). By use of a dominant negative TGFbetaII receptor, TGF-beta signaling was blocked, and its participation in the consequences of apoptotic cell stimulation was determined. The induction of TGF-beta itself could be attributed to exposed phosphatidylserine on the apoptotic cells, which therefore appears to drive the balanced inflammatory mediator responses. Arachidonic acid release, COX-2, and prostaglandin synthase expression were shown to be significantly dependent on the TGF-beta production. On the other hand, a requirement for TGF-beta was also shown in the inhibition of thromboxane synthase and thromboxanes, of 5-lipoxygenase and sulfidopeptide leukotrienes, as well as of inducible nitric-oxide synthase and NO. TGF-beta-dependent induction of arginase was also found and would further limit the NO generation. Finally, apoptotic cells stimulated production of 15-lipoxygenase and 15-hydroxyeicosatetraenoic acid, a potentially anti-inflammatory pathway acting through peroxisome proliferator-activated receptor-{gamma}, and lipoxin A4 production, which were also up-regulated by a TGF-beta-dependent pathway in this system. These results strongly suggest that the apoptotic cell inhibition of pro-inflammatory mediator production is pleiotropic and significantly dependent on the stimulation of TGF-beta production.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Recognition and clearance of apoptotic cells by phagocytes play pivotal roles in development, maintenance of tissue homeostasis, control of the immune response, and resolution of inflammation (1, 2). Apoptotic cells are removed by professional phagocytes, members of the mononuclear phagocyte system such as macrophages and immature dendritic cells, or by nonprofessional phagocytes such as fibroblasts, endothelial, epithelial, smooth muscle, or stromal cells (3). Uptake of the apoptotic cell is by a specialized and highly conserved form of phagocytosis termed efferocytosis (4, 5). As a cell becomes apoptotic, it is generally removed in situ by near-neighbor cells or macrophages in a quiet, almost invisible fashion, i.e. the process does not induce a local tissue reaction. In fact, recognition and removal of apoptotic cells are normally both anti-inflammatory and anti-immunogenic (6–9).

The interaction and recognition are triggered by surface changes on the apoptotic cells. Two widely distributed surface ligands on apoptotic cells are phosphatidylserine (PS)4 (10, 11) and calreticulin (5), which become associated in patches together on the cell surface. Indirect effects of the collectin family of molecules or direct action of calreticulin leads to stimulation of low density lipoprotein receptor-related protein (LRP) on the phagocytosing cell (5, 12). However, LRP activation seems to induce production of pro-inflammatory mediators (13). On the other hand, there is considerable evidence to implicate PS as the main stimulus for the anti-inflammatory or anti-immunogenic effects (6–8, 14–16). We suspect that these two stimuli, acting through different signaling pathways, are balanced, with a normal bias toward the anti-inflammatory. Unfortunately, the receptor(s) that recognizes PS (PS recognition structures, PSRS) that is responsible for this effect is unknown, although it does seem to distinguish between stereoisomeric forms of the phosphoserine head group (10, 14) and does seem to react with an activating IgM antibody mAb217 (17) whose binding is blocked by PS. The antibody binds to and activates cells and mimics exactly the effects of PS on apoptotic cells in contributing to uptake and on the generation of anti-inflammatory mediators (7, 18), and it has been used here along with apoptotic cells to stimulate macrophages for production or suppression of eicosanoids.

A major anti-inflammatory mediator induced in response to apoptotic cells, mAb217, or PS liposomes is TGF-beta (6, 8, 16). Blockade of TGF-beta has been shown to reverse the suppressive effects of apoptotic cells or PS in vivo on either inflammation or adaptive immunity (7, 8). On the other hand, earlier studies also suggested induction of other candidates such as IL-10 (19), PGE2 (6), and even platelet-activating factor (6), although the last two can have both pro- or anti-inflammatory effects. The ability of apoptotic cell recognition to alter the production of eicosanoids had first been noted for thromboxane (20, 21) and exemplified by roles for TGF-beta and prostaglandins in the apoptotic cell enhancement of Trypanosoma cruzi growth in macrophages (22). Therefore, a key issue is whether apoptotic cell-induced TGF-beta, acting in an autocrine/paracrine fashion, mediates the alterations in eicosanoid generation. By use of a dominant negative TGF-beta receptor construct, we have been able to show that apoptotic cells stimulate via their induction of active TGF-beta, a coordinate production of generally anti-inflammatory and simultaneous inhibition of generally pro-inflammatory, eicosanoids. The effect is mediated by effects on the synthases for these mediators. Additional coordinate effects were seen on related proteins, including iNOS, which was down-regulated, and PPAR{gamma} or arginase, which were induced, i.e. combining to reduce NO production and also potentially in keeping with the anti-inflammatory balance.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies and Reagents—TGF-beta was purchased from R&D Systems. Lipopolysaccharide (LPS, Escherichia coli 0111:B4) was from List Biological Laboratories, Inc. Recombinant murine interferon-{gamma} was from BD Biosciences. Human factor Va was from Hematologic Technologies Inc. Cyclosporin A and protease inhibitor mixture set I were from Calbiochem. Anti-arginase 1 antibody was from Santa Cruz Biotechnology. Antibodies against COX-2, human PGD-2 synthase, murine PGE-1 synthase 1, PGI synthase, thromboxane synthase, 5-LO, 15-LO II, PPAR{gamma}1, iNOS, and indomethacin were from Cayman Chemical. Anti-beta-actin antibody was from Cell Signaling. mAb217 is an IgM monoclonal antibody that was originally raised against PS-recognizing macrophages. It was obtained from concentrated hybridoma supernatants. Unfortunately, attempts to label, fragment, or convert the antibody to an IgG isotype have universally resulted in loss of activity. [5,6,8,9,11,12,14,15-3H]Arachidonate acid (200 Ci/mol) was from American Radiolabeled Chemical. Lipofectamine Plus reagent was from Invitrogen.

Induction of Apoptotic Cells—Jurkat T cells were exposed to UV irradiation at 254 nm for 10 min. Jurkat T cells were cultured in RPMI 1640 with 10% fetal calf serum (Gemini Bio-Products) for 3 h at 37°C in 5% CO2. The cells were generally ≥80% apoptotic by nuclear morphology and maintained intact cell membranes that excluded trypan blue (6).

Cell Culture, Stimulation and Measurement of Pro-inflammatory Mediators by ELISA—Murine peritoneal macrophages were obtained from BALB/c mice 4 days after intraperitoneal injection of 1 ml of thioglycollate. Murine RAW 264 cells were obtained from the American Type Culture Collection. RAW-V and RAW-TbetaRII were made by stable transfection of empty vector and truncated TGF-beta receptor II constructs, respectively. The cells (1 x 106 cells/well) were plated in each well of a 24-well tissue culture plate and were cultured in DMEM supplemented with 10% heat-inactivated endotoxin-free fetal bovine serum, 2 mML-glutamine, 100 µg/ml streptomycin, and 100 units/ml penicillin under a humidified 5% CO2 atmosphere at 37 °C for 24 h. Then the cells were cultured in serum-free DMEM in the absence or presence of LPS, IFN-{gamma}, factor Va, or cyclosporin A for 18 h with 3 x 106 apoptotic Jurkat cells (apoJ), viable Jurkat cells (ViableJ), 50 µg/ml mAb217, 50 µg/ml control isotype IgM, or 100 µM liposomes (containing 30:70 molar ratios of PS:PC or PC alone). The supernatants were collected and measured for TGF-beta, TNF-{alpha}, PGE2, PGF1{alpha}, 15-HETE, LXA4, and leukotrienes by ELISA according to the manufacturer's instructions (ELISA TECH, Aurora, CO). In some experiments, to inhibit pro-inflammatory mediators, the macrophages were treated with stimuli cited before and 100 ng/ml LPS or LPS plus 40 units/ml IFN-{gamma}.

NO Production Assay—NO levels produced by RAW 264 cells, RAW-TbetaRII cells, and murine peritoneal macrophages were measured by reducing the nitrate accumulated over 18 h to nitrite with nitrate reductase (23) and measuring the nitrite concentration by the method of Green et al. (24). The nitrite concentrations were quantified by using a double three-point standard curve of NaNO2 concentrations (in a linear range between 1 and 80 µM).

Measurement of Arachidonate Release—Arachidonic acid release was measured as described (25). Briefly, the cells were cultured in 24-well plates to incorporate arachidonate by incubation for 24 h in 1 ml of DMEM containing 10% fetal bovine serum and [3H]arachidonate (1 µCi/ml) for 24 h. Then the cells were washed and incubated in serum-free DMEM containing 0.1% human serum albumin and stimulated with LPS or apoJ or ViableJ or mAb217 or isotype control IgM. The medium was removed 2 h after stimulation and centrifuged at 500 x g for 10 min, and the amount of radioactivity (arachidonate and arachidonate products) in the supernatant was determined.

Transient Cell Transfection and Reporter Gene Assay—The p3TP-luc (26) luciferase reporter gene construct was transfected into RAW-TbetaRII and RAW-V cells using Lipofectamine Plus reagent according to the manufacturer's instructions. pSV-beta-galactosidase vector (Promega) was co-transfected as an internal control to measure differences in transfection efficiency. Luciferase and beta-galactosidase activities were measured 18 h after TGF-beta stimulation using the luciferase assay system (Promega) and Galacto-Light (Tropix), respectively.

Immunoblotting Analysis—Immunoblotting analysis was carried out as described previously with some modification (27). Briefly, cells (3.0 x 105 cells/well) were plated in each well of a 12-well tissue culture plate and incubated overnight. Following stimulation, the cells were lysed in lysis buffer (20 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM dithiothreitol, 0.5% Triton X-100 and 1x protease inhibitor mixture set I), resolved on 10% SDS-PAGE, and blotted onto nitrocellulose membranes. The membranes were probed with primary antibodies at 4 °C overnight and incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Proteins were visualized by enhanced chemiluminescence (Amersham Biosciences) according to the manufacturer's instructions. Equal loading of proteins in each lane was confirmed by Ponceau S staining or re-probed with corresponding antibodies against the native proteins (28) or beta-actin. The results shown are representative of at least three separate experiments.


Figure 1
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  		FIGURE 1.
Apoptotic cells or mAb217 stimulate production of TGF-beta and suppress LPS-induced TNF-{alpha}, NO, and iNOS. A, thioglycollate-elicited mouse peritoneal macrophages or RAW 264 macrophages were incubated in the presence of LPS (100 ng/ml), mAb217 (50 µg/ml), isotype IgM (50 µg/ml), apoptotic (apoJ), or viable Jurkat (ViableJ) cells (3 x 106 cells/ml) for 18 h. TGF-beta in the conditioned medium was analyzed by ELISA. *, significantly different from controls. B, RAW 264 macrophages were preincubated with different concentrations of factor Va for 30 min and then incubated with viable or apoptotic Jurkat cells for 18 h. *, significantly different from controls. #, significantly different from apoJ alone. C, RAW 264 macrophages were incubated with LPS, LPS plus PS liposomes (100 µM, 30:70 ratio of PS: PC), LPS plus PC liposomes (100 µM), and PS or PC liposomes alone for 18 h (upper panel). RAW 264 macrophages were incubated with indicated concentrations of cyclosporin A (CsA) or cyclosporin A plus PS liposome for 18 h (lower panel). #, significantly different from LPS or cyclosporin A alone. *, significantly different from controls (C). D and E, peritoneal macrophages or RAW 264 macrophages were preincubated with mAb217, isotype IgM, apoJ, or ViableJ for 30 min and then stimulated with LPS (100 ng/ml) plus IFN-{gamma} (40 units/ml) for 18 h. TNF-{alpha} or NO (measured as nitrite) concentrations were analyzed in the conditioned medium. #, significantly different from controls. *, significantly different from LPS/IFN-{gamma}. (E, lower panel) iNOS protein levels in the total cell lysate were analyzed by Western blot.

 
Statistical Analysis—All data are presented as means ± S.E. from three or more separate experiments. The means were analyzed using analysis of variance for multiple comparisons. When analysis of variance indicated significance, the Tukey-Kramer honestly significant difference test for all pairs was used to compare groups. All data were analyzed using JMP statistical software (version 5; SAS Institute) for the Macintosh computer.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Apoptotic Cells or Antibody to PSRS on Murine Macrophages Stimulate Production of TGF-beta and Concomitant Blockade of LPS-induced TNF-{alpha}, NO, and iNOS—Murine macrophages (peritoneal or RAW 264, 1.0 x 106 cells/ml) were stimulated with LPS (100 ng/ml) as positive control or mAb217 (50 µg/ml) or apoptotic Jurkat T cells (3 x 106 cells/ml) for 18 h. These stimuli each induced TGF-beta production in both types of macrophages (Fig. 1A). Isotype control IgM or viable Jurkat T cells were inactive. These data are in accordance with previous reports (6, 16). The stimulation of TGF-beta production by apoptotic cells in this system was blocked by preincubation of the targets with the PS-binding protein factor Va (Fig. 1B) as had been shown earlier with annexin V (29). PS liposomes also stimulated the production of TGF-beta but less efficiently. This is probably because the presentation to the PSRS from PS exposed on the apoptotic cell is from a quite different environment compared with a liposome. However, PS liposomes increased the production of TGF-beta in the presence of LPS or cyclosporin A (Fig. 1C). These findings suggest that PS liposomes themselves may up-regulate TGF-beta translation when TGF-beta message has been induced by other stimuli (17, 30). Classically activated macrophages (LPS and IFN-{gamma} stimulation) exhibit release of TNF-{alpha} and NO as well as up-regulation of iNOS. As shown in Fig. 1, D and E, these three responses to stimulation with LPS and IFN-{gamma} were inhibited by exposure of the macrophages to apoptotic cells or mAb217. Previous studies implicated the TGF-beta produced, in the suppression of TNF-{alpha} induction, and might be expected to serve the same role for suppression of iNOS and NO.


Figure 2
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  		FIGURE 2.
Transfection of truncated TGFbetaRII into RAW 264 cells reverses the suppression of TNF-{alpha}, NO, iNOS, and arginase 1 up-regulation induced by apoptotic cells or PSRS stimulation. A, RAW macrophages transfected with truncated TGF-beta receptor II (RAW-TbetaRII) and empty vector (RAW-V) were transiently co-transfected with 3xPT-luc and pSV-beta-galactosidase constructs. After 48 h, the cells were incubated in the presence of TGF-beta (10 ng/ml) for 18 h. The luciferase assays, which were normalized to beta-galactosidase, are expressed as relative luciferase unites. #, significantly different from controls. *, significantly different from RAW-V cells stimulated with TGF-beta. B, RAW-V or RAW-TbetaRII cells were incubated in the presence of LPS, mAb217, isotype IgM, apoJ, or ViableJ for 18 h. #, significantly different from controls. *, significantly different from RAW-V cells. C and D, RAW-V or RAW-TbetaRII cells were incubated with the stimuli for 30 min and then with LPS plus IFN-{gamma} for 18 h. TNF-{alpha} or NO were measured in the conditioned medium. #, significantly different from LPS/IFN-{gamma}.*, significantly different from RAW-V cells. E and F, iNOS or arginase protein levels in the total cell lysate were analyzed by Western blot.

 
To demonstrate this presumed requirement for TGF-beta in the suppression, a dominant negative form of the TGFbetaRII was employed. Transfection of RAW 264 cells with this construct was shown to block the ability of TGF-beta to signal for 3xPT-luc reporter (which contains three consecutive 12-O-tetradecanoylphorbol-13-acetate-response elements and a portion of the plasminogen activator inhibitor (PAI-1) promoter region) gene activation (Fig. 2A). Because TGF-beta can induce its own synthesis, the effect of the dominant negative receptor was also examined on the production of TGF-beta itself after stimulation with apoptotic cells, mAb217, or LPS. As shown in Fig. 2B, this treatment blocked 60–70% of the TGF-beta produced by each of the stimuli, i.e. supporting an additional autocrine/paracrine effect of TGF-beta on its own induction in these systems.

Transfection of the truncated TGFbetaRII was found to completely reverse the suppression of TNF-{alpha} and NO production caused by apoptotic cells or PSRS stimulation and also restored the up-regulation of the iNOS protein (Fig. 2, C–E). In keeping with the suppression of NO production by blocking up-regulation of iNOS, exposure of macrophages to apoptotic cells or PSRS stimuli also led to increases in intracellular levels of arginase 1 (Fig. 2F), which could further reduce the production of nitric oxide.

Macrophages with Truncated TGFbetaRII Are Defective in Prostaglandin Production and Prostaglandin Synthase Expression in Response to Stimulation with Apoptotic Cells or LPS—In the original studies of anti-inflammatory effects of apoptotic cells, PGE2 was also shown to be generated, and it too seemed to play a role in suppression of inflammatory mediators (27). Accordingly, we next examined the effect of apoptotic cells and stimulation with mAb217 on induction of potentially anti-inflammatory prostaglandins as well as the role of TGF-beta in their regulation. The original studies did not address the probable induction of PGI2 (detected as PGF1{alpha}) along with PGE2, and accordingly, this was included in the analysis. Supernatants from the cell culture were collected 18 h after stimulation and analyzed for PGE2 and PGF1{alpha}. The cell lysates were collected, and the levels of synthases for PGE2 (PGES1), PGD2, and PGI2 were determined by Western blotting. As expected, the two stimuli induced production of PGE2 and PGF1{alpha} starting at 2 h or earlier and extending out to 18 h of incubation (Fig. 3, A–C). Importantly, they also increased the intracellular levels of the prostaglandin synthases (Fig. 3D).

It has been reported previously that TGF-beta can induce prostaglandin production (22, 31, 32), and in data not shown, the direct addition of active TGF-beta to the macrophage cultures did stimulate production of PGE2 and PGF1{alpha}. When the macrophage response to TGF-beta was blocked with the dominant negative receptor, induction of PGE2 and PGF1{alpha} by either apoptotic cells or mAb217 was prevented (Fig. 3, A and B). Interestingly, LPS-induced PGE2 and PGF1{alpha} were also reduced by about 80%. We suspect that the lack of complete blockade with this stimulus reflects the possible use of alternative pathways not involving TGF-beta. In keeping with the data on the prostaglandins themselves, the truncated receptor also reduced up-regulation of the synthases (Fig. 3D), although not to as great an extent as seen for the secreted prostaglandins.

These prostaglandins are lipid mediators that like TGF-beta have been reported to have pro- or anti-inflammatory properties in different circumstances. For example, we earlier showed PGE2 to decrease TNF-{alpha} production from macrophages (6). A possible contributory effect of prostaglandins themselves to TGF-beta production is depicted in Fig. 3E, wherein indomethacin was shown to be able to reduce the amount of TGF-beta produced in response to apoptotic cells and mAb217 (see also Ref. 6). This, along with the autostimulation of TGF-beta by TGF-beta (see above), further shows the extensive feedback responses inherent in these systems.


Figure 3
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  		FIGURE 3.
Prostaglandin production and prostaglandin synthase expression in response to apoptotic cells or mAb217 stimulation is TGF-beta-dependent. A and B, RAW-V or RAW-TbetaRII cells were incubated in the presence of LPS, mAb217, isotype IgM, apoJ, or ViableJ for 18 h. PGE2 or PGF1{alpha} concentrations in the conditioned medium were analyzed by ELISA. *, significantly different from RAW-V. C, PGE2 and PGF1{alpha} time course. RAW cells were incubated in the presence of the stimuli, and the supernatant was collected after 30 min and 2, 4, 8, and 18 h. D, PGE-1, human PGD and PGI synthase protein levels in the total cell lysate were analyzed by Western blot. E, RAW cells were stimulated with mAb217, isotype IgM, apoJ, or ViableJ in the presence or absence of indomethacin (Indo) for 18 h. TGF-beta concentration was analyzed by ELISA.

 
TGF-beta-dependent Suppression of Thromboxane Synthesis—Early studies of responses to apoptotic cells showed that thromboxane production was decreased (6, 20, 21). This suggests coordinate up-regulation of potentially anti-inflammatory prostanoids along with down-regulation of pro-inflammatory thromboxane and raises the question of whether TGF-beta is responsible for both effects. Because macrophages do not produce TXA2 spontaneously, and did not do so at any time after incubation with apoptotic cells or mAb217 alone (data not shown), they were activated with LPS to demonstrate the suppressive effect of co-stimulation with either apoptotic cells or mAb217 (Fig. 4A). Almost complete inhibition of TXA2 (measured as thromboxane B2) was seen, and transfection of the macrophages with the dominant negative TGF-beta receptor reversed this inhibition. Because LPS also induces TGF-beta production, enhancement of the LPS effect on thromboxane production might have been seen if this TGF-beta was blocked. However, this was not observed, probably because the generation of active TGF-beta following LPS occurred after the majority of the thromboxane had already been produced. Once again, the effect of the TGF-beta appeared to be at the level of the synthase. Thromboxane synthase levels in the macrophages were suppressed by apoptotic cells and mAb217, but not after transfection of the truncated TGF-beta receptor (Fig. 4B).

Stimulation of Macrophages with mAb217 or Apoptotic Cells Induced COX-2 Expression and Arachidonic Acid Release through TGF-beta-dependent Signaling—The demonstrated reciprocal effect of TGF-beta on the prostanoid synthases raised the possibility that the induction of PGE2 and PGI2 reflected a diversion of the precursor PGH2 from utilization by thromboxane synthase. However, because the prostaglandins were increased directly in macrophages that did not express the thromboxane synthase, it seemed likely that the apoptotic cells also led to increased levels of one or other PGH synthases (COX enzymes) and thereby increased production of PGH2. No evidence was found for altered amounts of COX-1 (data not shown), but both apoptotic cells and mAb217, as well as LPS as expected, did increase the intracellular amounts of COX-2 (Fig. 5A). In keeping with the theme of this study, macrophages expressing the truncated TGF-beta receptor did not show up-regulation of COX-2 in response to the apoptotic cells and markedly reduced that stimulated by LPS.

To initiate eicosanoid production, a source of free arachidonate must be available and, presumably, must be initiated by the apoptotic cell or mAb217 stimulus. This is shown in Fig. 5B where free [3H]arachidonate (including its metabolites) was measured in the supernatant after previous incorporation into macrophage phospholipids before stimulation. In this case, blocking the TGF-beta effects with the dominant negative receptor reduced the amounts of arachidonate released by about 50% in the case of all three stimuli. This presumably reflects either a timing issue as noted for the LPS-induced thromboxane or the availability of preformed phospholipases that were stimulated by either the LPS or PSRS engagement in addition to an effect from TGF-beta. TGF-beta has been reported to induce prostaglandin production (22, 31, 32), which would necessitate its ability to initiate released arachidonic acid, an effect that was shown here in Fig. 5B.


Figure 4
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  		FIGURE 4.
TGF-beta-dependent suppression of thromboxane synthesis induced by apoptotic cells or mAb217 stimulation. A, RAW-V or RAW-TbetaRII cells were incubated with the stimuli for 30 min and then with LPS (100 ng/ml) for 18 h. Thromboxane (measured as thromboxane B2) concentrations in the conditioned medium were analyzed using ELISA. #, significantly different from LPS. *, significantly different from RAW-V cells. B, thromboxane synthase levels in the total cell lysate were analyzed by Western blot.

 


Figure 5
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  		FIGURE 5.
TGF-beta-dependent induction of COX2 expression and arachidonic acid release by apoptotic cells or mAb217 stimulation. A, RAW-V or RAW-TbetaRII cells were incubated with the stimuli for 18 h. COX-2 levels in the total cell lysate were analyzed by Western blot. B, RAW-V or RAW-TbetaRII cells were labeled with [3H]arachidonate (1 µCi/ml) for 24 h and then stimulated for 2 h. Arachidonic acid and products released in the supernatant were analyzed as described under "Experimental Procedures." *, significantly different from RAW-V cells.

 
Acting through TGF-beta, Apoptotic Cells or mAb217 Decreased LPS-enhanced 5-Lipoxygenase and Leukotrienes but Increased 15-LO, 15-HETE, and LXA4 as Well as PPAR{gamma}—The pattern of TGF-beta induction of potentially anti-inflammatory eicosanoids, but the suppression of pro-inflammatory eicosanoids, was further explored by examining the effects on lipoxygenases and leukotrienes. Our earlier study (6) had shown suppression of leukotriene release from macrophages by apoptotic cells. Stimulation of macrophages with apoptotic cells or mAb217 by themselves did not induce cysteinyl leukotrienes at 18 h and suppressed that induced by priming with LPS (Fig. 6A). However, a time course study (Fig. 6B) did show an early production at 2 h but none subsequently. Similarly, LTB4 was seen only 2 h after stimulation, after this time the production was down-regulated (data not shown). Examination of the key upstream enzyme 5-lipoxygenase revealed, as expected, that its levels were increased after priming with LPS but that this did not occur in the presence of the apoptotic cells or mAb217 (Fig. 6C). In the presence of the truncated TGF-beta receptor, the LPS-primed increase in levels of 5-LO was no longer prevented (Fig. 6C). We attribute the early induction of leukotrienes to an effect of endogenous 5-LO acting on early released arachidonate before the TGF-beta effect has time to kick in.

By contrast, when 15-LO was examined, the apoptotic cell stimulus directly increased intracellular levels of the enzyme and production of 15-HETE (Fig. 6, D–F). The 15-HETE production was seen 4–8 h after stimulation and peaked at 18 h (Fig. 6F). Once again, the induction by apoptotic cells or mAb217 was blocked in the presence of the truncated TGF-beta receptor. On the other hand, TGF-beta did not appear to play a significant role in the induction of 15-LO or 15-HETE by LPS. Products of 15-LO have been suggested to participate in LXA4 production and activation of PPAR{gamma} (33, 34), which may also have anti-inflammatory effects in macrophages (35–37). Accordingly, we also examined the effect of apoptotic cells and mAb217 to induce LXA4 and alter the levels of PPAR{gamma}. As shown in Fig. 7, A and B, both stimuli initiated LXA4 production and increased the amounts of PPAR{gamma} protein in the cells, as did direct addition of TGF-beta. This supports a possible additional anti-inflammatory effect of apoptotic cells via LXA4 and/or activation of PPAR{gamma}.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Apoptotic cells are known to induce an anti-inflammatory and anti-immunogenic response, mediated in part by their induction of active TGF-beta in responding cells. Here we show that the effect of the apoptotic cells is to drive a complex coordinated inhibition of potentially inflammatory mediators along with induction of potentially anti-inflammatory molecules in macrophages that are orchestrated by the production of TGF-beta. By studying the responses induced by apoptotic cells in macrophages that are unresponsive to TGF-beta by virtue of their transfection with a dominant negative TGF-beta receptor, the role of this important mediator on a wide variety of eicosanoids, nitric oxide, and related molecules was delineated. Thus, the earlier demonstrations that apoptotic cells induced the production of PGE2 but suppressed thromboxane (6, 20) were both shown to be due to the effects of TGF-beta, and these observations extended to other potentially pro- and anti-inflammatory arachidonate metabolites. It should be noted that the prostanoids PGE2, PGI2, and PGD2 are known to exhibit both pro- and anti-inflammatory actions, in part for PGE2, depending upon the receptors that are engaged (38). Similar pleomorphic effects should be noted for NO. In our earlier study (6), we showed that PGE2 suppressed LPS-induced inflammatory mediators from macrophages (6), and in the data reported here, prostanoid production also showed a feedback enhancement of TGF-beta production. Thus, in this limited context, we see these prostanoids as potentially anti-inflammatory, and their induction as contributing to the overall anti-inflammatory consequences of apoptotic cell recognition.


Figure 6
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  		FIGURE 6.
Apoptotic cell or mAb217 stimulation decreases LPS-induced 5-lipoxygenase and leukotrienes but increases 15-lipoxygenase and 15-HETE through TGF-beta. A, RAW 264 macrophages were pre-incubated with apoJ or mAb217 for 30 min and then stimulated with or without LPS for 18 h. Leukotriene production in the conditioned medium was analyzed by ELISA. *, significantly different from LPS alone. B, time course of leukotriene production. C, RAW-V or RAW-TbetaRII cells were incubated with mAb217, isotype IgM, apoJ, or ViableJ for 30 min and then with LPS for 18 h. 5-LO levels in the total cell lysate were analyzed by Western blot. D–F, RAW-V or RAW-TbetaRII cells were incubated with the stimuli or TGF-beta (C, 10 ng/ml) for 18 h. D, 15-LO levels in the total cell lysate were analyzed by Western blot. E and F, 15-HETE production in the conditioned medium was analyzed by ELISA. *, significantly different from RAW-V cells.

 


Figure 7
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  		FIGURE 7.
Apoptotic cells or mAb217 increase LXA4 production and PPAR{gamma}. A, RAW-V or RAW-TbetaRII cells were incubated with stimuli or TGFbeta for 18 h, and PPAR-{gamma} levels in the total cell lysate were analyzed by Western blot. B, RAW 264 macrophages were incubated with the stimuli or TGFbeta for 18 h. LXA4 concentration was measured in supernatant by ELISA. *, significantly different from RAW-V.

 
The observations required the demonstration of TGF-beta production in response to the apoptotic cells, shown earlier by numerous investigators and confirmed herein. A number of ligands have been demonstrated on apoptotic cells that can interact with a number of "receptors" on responding cells, in this case macrophages. Additionally there are a large group of "bridge" molecules (39) thatcanlinktheapoptoticcellligandsto the receptors. We have suggested that two important ligands are phosphatidylserine and calreticulin. The latter, as well as the collectin family of bridge molecules (40), has been suggested to interact with LRP as a receptor and, in isolation, seems to induce a more pro-inflammatory response (5, 13). On the other hand, PS and its receptors and possibly some or all of its bridge molecules appear to induce the anti-inflammatory effects and, in most cases, to act in a dominant fashion in the normal response to apoptotic cells. Necrotic cells are usually thought to be pro-inflammatory (9, 15) and may have reversed this PS-driven dominance. Other studies that have suggested that apoptotic cells can in some circumstances act in a pro-inflammatory fashion may also reflect variations in balance between pro-inflammatory (e.g. LRP) versus anti-inflammatory (e.g. PS-driven) responses.

In the studies reported here, we show that the TGF-beta induction by apoptotic cells was dependent on exposed PS by blockade with the PS-binding protein factor Va, which has the advantage over the more usually employed annexin V by binding in physiologic concentrations of calcium. Although direct stimulation of TGF-beta by PS-containing liposomes was weak, these agents readily enhanced TGF-beta production to other stimuli. The amounts of TGF-beta measured by ELISA only represents that in the supernatant and is therefore relatively insensitive for a molecule that binds to surfaces and cell membranes. Early in vivo demonstrations of PS liposome effects were also relatively weak in comparison with apoptotic cells (8) and, we suspect, may reflect issues of presentation and avidity.

The other approach employed here was to examine the effect of mAb217, an IgM-activating antibody that is suspected of binding to an as yet unidentified receptor for PS on responding cells. Its ability to bind all cells that respond to PS-exposing apoptotic cells, to be blocked itself by pretreatment of the cells with L- but not D-phosphatidylserine, and its close mimicking of the activation induced by PS-exposing apoptotic cells supports this contention. In all cases in this study, mAb217 and apoptotic cells behaved identically. The implication is that recognition of PS drives the production of TGF-beta and the downstream anti-inflammatory responses reported herein.

The eicosanoids are derived from metabolism of arachidonate through a number of pathways. In this study we focused on prostanoid synthesis through PGH synthases (COX) and downstream prostaglandin and thromboxane synthases as well as through lipoxygenases and their downstream products of leukotrienes and HETES. Apoptotic cell and TGF-beta induction of PGE2 and PGI2 were shown. The effect appeared to be the up-regulation of the respective synthases PGES and PGIS whose protein levels were increased in response to the apoptotic cells and blocked in the presence of the dominant negative TGF-beta receptor. Likewise, PGD synthase protein levels were up-regulated by the apoptotic cells and mAb217 but not in the absence of TGF-beta effects. By contrast, the pro-inflammatory eicosanoid thromboxane was suppressed. In this case the resting macrophages or those stimulated with apoptotic cells alone did not produce thromboxane or exhibit significant levels of its synthase. When stimulated to induce thromboxane synthase by LPS or LPS and IFN-{gamma}, however, the apoptotic cells or mAb217 suppressed the up-regulation and the mediator production, again in a TGF-beta-dependent fashion.

These effects on prostanoid balance implied an up-regulation of both COX and of sources of substrate, namely arachidonic acid, and both increases in COX2 (but not COX1) and released arachidonate were demonstrated in response to apoptotic cells. The effect on COX2 was also shown to be dependent on TGF-beta (41, 42) and at least part of the arachidonate release. TGF-beta itself also induced liberation of arachidonate. Although not explored directly herein, it seems reasonable to assume that the apoptotic cells and/or TGF-beta induce activation of existing phospholipases 2 and that the TGF-beta may also initiate some up-regulation of these that contributes to greater and/or more prolonged release of the arachidonate.

Our earlier study had suggested that apoptotic cells also suppressed the production of potentially pro-inflammatory sulfidopeptide leukotrienes (6). Here we show that levels of the upstream enzyme 5-lipoxygenase, as well as of the leukotrienes themselves, were also suppressed by the apoptotic cells and that this too was reversed in the absence of TGF-beta signaling. The decrease in 5-lipoxygenase levels noted here are at odds with studies showing enhanced production in response to TGF-beta during macrophage maturation under the influence of vitamin D3 (43). We therefore suspect a discordant effect of TGF-beta on maturing versus mature macrophages. Intriguingly, and in keeping with the coordinated anti-inflammatory effects, 15-lipoxygenase was up-regulated, and its stable product 15-HETE was increased by the apoptotic cells or mAb217, again because of TGF-beta. Products of 15-HPETE are generally thought to be anti-inflammatory (44–47), and some may possibly achieve this effect via activation of PPAR{gamma} (34). Accordingly, we also addressed the effect of the apoptotic cells and TGF-beta pathways on LXA4 production and the up-regulation of PPAR{gamma}, and we showed that those too were increased.

The other pathway and mediator addressed in this study was the potentially immunoregulatory and broad spectrum signaling molecule NO. Apoptotic cells and mAb217 suppressed the generation of NO and the up-regulation of iNOS. TGF-beta is well known to inhibit production of iNOS (48–50); therefore, the TGF-beta dependence of this was expected and shown by the dominant receptor approach. Intriguingly, arginase, which reduces the substrate for NOS enzymes, was reciprocally up-regulated by the apoptotic cells, again via TGF-beta. This would provide an additional brake on the NO generation by macrophages responding to apoptotic cells.

The results indicate a complex effect of apoptotic cells acting through release of TGF-beta to up-regulate generally anti-inflammatory mediators and inhibit the production of pro-inflammatory molecules. The systems employed here support an effect on synthesis of the various enzymes involved in generating these mediators, i.e. acting on transcription, translation, or both. However, we cannot exclude additional effects on metabolism or secretion of the mediators acting in the shorter term. In response to apoptotic cells, macrophages appear to release preformed TGF-beta almost immediately (30–60 min (8, 16)) followed by new synthesis and more prolonged generation of the active molecule. Combined, this would achieve a fairly rapid and then prolonged effect on the inflammatory state of the cell. However, it should be noted that the immediate generation of free arachidonate in response to apoptotic cells might lead to generation of potentially pro-inflammatory eicosanoids by enzymatic pathways constitutively present in the cell (e.g. 5-LO and leukotriene synthases) before the slower effects of TGF-beta had a chance to kick in (see for example Refs. 51–54). Given the pluripotentiality and plasticity of this cell type, we are generally skeptical of the concept of stable macrophage "phenotypes" (3). Nevertheless, it should also be noted that some of the effects of apoptotic cell exposure mimic those seen in the so-called alternatively activated macrophage (55, 56), thus raising intriguing questions regarding how long these effects persist following the interaction with apoptotic cells and/or the consequences of prolonged exposure as might be experienced in resolving inflammatory response in vivo.


   FOOTNOTES
 
* This work was supported in part by National Institutes of Health Grants HL81151 and HL67671. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Both authors contributed equally to this work. Back

2 Recipient of Long Term Fellowship LT-00606-2002 from the Human Frontier Science Program. Back

3 To whom correspondence should be addressed: Program in Cell Biology, Dept. of Pediatrics, National Jewish Medical and Research Center, 1400 Jackson St., Denver, CO 80206. E-mail: hensonp{at}njc.org.

4 The abbreviations used are: PS, phosphatidylserine; PSRS, phosphatidylserine recognition structures; TNF-{alpha}, tumor necrosis factor-{alpha}; TGF-beta, transforming growth factor-beta; LPS, lipopolysaccharide; IFN-{gamma}, interferon-{gamma}; COX, cyclooxygenase; PG, prostaglandin; NO, nitric oxide; LO, lipoxygenase; PPAR{gamma}, peroxisome proliferator-activated receptor-{gamma}; iNOS, inducible nitric-oxide synthase; LXA4, lipoxin A4; mAb, monoclonal antibody; ELISA, enzyme-linked immunosorbent assay; DMEM, Dulbecco's modified Eagle's medium; LRP, low density lipoprotein receptor-related protein. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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