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The Journal of Immunology, 2003, 171: 1507-1514.
Copyright © 2003 by The American Association of Immunologists

Murine Macrophages Produce Secretory Leukocyte Protease Inhibitor During Clearance of Apoptotic Cells: Implications for Resolution of the Inflammatory Response 1

Chikako Odaka2,*, Toshiaki Mizuochi*, Jingxuan Yang{dagger} and Aihao Ding{dagger}

* Department of Safety Research on Blood and Biological Products, National Institute of Infectious Diseases, Tokyo, Japan; and {dagger} Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, NY 10021


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Macrophage-derived secretory leukocyte protease inhibitor (SLPI) can be induced locally as well as systemically in response to microbial products such as LPS and lipotechoic acid. It is not known whether phagocytosis of apoptotic cells, an essential function of macrophages, can regulate expression and secretion of SLPI. In this study, we report that exposure of peritoneal macrophages of BALB/c mice or murine macrophage cell lines RAW264.7 and J774.1 to apoptotic target cells induced an elevation in SLPI secretion. Secreted SLPI retained its antichymotrypsin activity. SLPI expression in thymuses from BALB/c mice that had been injected with anti-CD3 Ab to induce apoptosis of thymocytes was also elevated both at the mRNA and protein levels. Colchicine, a microtubular inhibitor, blocked the internalization of apoptotic cells by macrophages but not SLPI secretion, suggesting that surface recognition of apoptotic cells is sufficient for the induction of SLPI. Exposure of RAW264.7 cells to apoptotic CTLL-2 cells induced both SLPI and TNF-{alpha}, and addition of IFN-{gamma} inhibited SLPI but augmented TNF-{alpha} production. Transfection of either the secreted or a nonsecreted form of SLPI into RAW264.7 cells led to suppression of TNF-{alpha} production in response to apoptotic cells. Thus, macrophages secrete an increased amount of SLPI when encountering apoptotic cells, which may help to attenuate potential inflammation during clearance of these cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Cells that die in physiological contexts are removed rapidly by phagocytic cells, including macrophages (1). Macrophages are key players in maintaining immune homeostasis as well as resolving inflammation, which is often orchestrated by a number of bioactive products of macrophages in response to an everchanging physiological environment. These include pro- and anti-inflammatory cytokines (e.g., TNF-{alpha}, TGF-{beta}, and IL-10) in addition to nonprotein products (e.g., eicosanoids, reactive oxygen, and nitrogen intermediates). It is generally believed that uptake of apoptotic cells by phagocytes does not provoke the release of proinflammatory cytokines (2). In fact, uptake of apoptotic neutrophils by LPS-stimulated human monocyte-derived macrophages inhibited the production of TNF-{alpha} through the induction of TGF-{beta} (3, 4, 5). Apoptotic cells were also shown to modify cytokine production in LPS-activated monocytes, including an augmentation of IL-10 (6, 7). Thus, clearance of apoptotic cells by macrophages is accompanied by altered cytokine profiles that ensure an effective clearance without damaging bystander tissues.

Secretory leukocyte protease inhibitor (SLPI) 3 is an 11.7-kDa nonglycosylated protein originally found in epithelial cells at mucosal surfaces (8, 9). It was identified as a potent inhibitor of serine proteases, including neutrophil elastase, cathepsin G, and a chymotrypsin-like enzyme (8, 10, 11). Further investigation revealed that macrophages and neutrophils are rich sources of SLPI, and SLPI has been recognized to have antibacterial, antiviral, and anti-inflammatory properties. These include the prevention of HIV replication in monocytic cells (12, 13), the down-regulation of prostaglandin, and matrix metalloprotease synthesis by monocytes (14), and the inhibition of inflammatory lung injury caused by deposition of IgG-immune complexes (15), or of joint damage caused by bacterial cell wall-induced arthritis (16). We have previously found that serum SLPI is elevated in human sepsis and experimental endotoxemia (17), and that SLPI expression in murine macrophages is induced by LPS and lipotechoic acid (18, 19) but suppressed by IFN-{gamma} (18). We also showed that SLPI antagonizes LPS-induced NF-{kappa}B activation and TNF-{alpha} secretion (18), which may be due to the prevention of I{kappa}B degradation by SLPI (20).

Based on these findings, we hypothesized that macrophages might induce SLPI during phagocytosis of apoptotic cells, and that SLPI may participate in the maintenance of immune homeostasis. In this study, we found that macrophages secreted an increased amount of bioactive SLPI upon interaction with apoptotic cells. We also found that macrophages released TNF-{alpha} in response to apoptotic cells, and that overexpression of SLPI by transfection suppressed TNF-{alpha} releases during target cell clearance. The present study may provide insight into the mechanisms by which macrophages control the inflammatory response during phagocytosis of apoptotic cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents and supplies

Tissue culture dishes were obtained from Corning Glass Works (Corning, NY). Mouse IFN-{gamma} (protein concentration 1.1 mg/ml, sp. act. 5.2 x 106 U/mg) was obtained from Genentech (South San Francisco, CA). Oligonucleotide primers were from Sawady Technology (Tokyo, Japan). Anti-mouse CD3{epsilon} mAb (145-2C11) was purified from ascite fluid by protein A-affinity chromatography. Normal rabbit IgG and rabbit anti-mouse SLPI IgG (18) were purified by protein A-affinity chromatography. LPS (Escherichia coli O55B5) was from Difco (Detroit, MI). Zymosan A bioparticles opsonizing reagent was from Molecular Probes (Eugene, OR). All other reagents were purchased from Sigma-Aldrich (St. Louis, MO) except as specified below.

Injection of anti-CD3 Ab

Seven-week-old female BALB/c mice were injected i. p. with 50 µg of anti-CD3 Ab. At the indicated time, mice were sacrificed. Thymuses were isolated, immersed in OCT compound (Miles, Naperville, IL), rapidly frozen on dry ice, and sectioned at 5 µm for immunostaining. Alternatively, thymuses were excised and used for protein extraction or total RNA isolation.

Cell preparations and cell culture

Cells were maintained at 37°C in a humidified, 5% (v/v) CO2 atmosphere in RPMI 1640 medium (Sigma-Aldrich) supplemented with 2 mM L-glutamine, 50 µM 2-ME, 20 U/ml penicillin, 20 µg/ml streptomycin, and 10% heat-inactivated FBS (FCS, v/v; HyClone Laboratories, Logan, UT). J774.1 and RAW264.7 cells are monocyte-derived macrophage cell lines from BALB/c mice. For preparation of primary mouse macrophages, peritoneal exudate cells were collected from the peritoneal cavity of 9- to 12-wk-old BALB/c mice, washed with RPMI 1640 complete medium twice, and seeded into 10-cm culture dishes at 2 x 106 cells per dish. After incubation at 37°C for 2 h, nonadherent cells were removed, and macrophage purity of the final cell preparation (Mac-1-positive) was consistently >96% as determined by FACS analysis (data not shown). The viability of the cells was >96% as determined by trypan blue exclusion. Thymocytes from thymus glands of 6- to 7-wk-old mice were seeded into 24-well culture plates at 5 x 106 cells/well, and treated with 1 µM dexamethasone at 37°C for 6 or 20 h to generate early or late apoptotic cells, respectively. Membrane integrity and the accessibility of phosphatidylserine of apoptotic target cells were assessed cytofluorometrically by staining with propidium iodide (PI) and FITC-conjugated annexin V. Populations of early apoptotic thymocytes were 60% annexin V-positive and 7% PI-positive, while those of late apoptotic thymocytes were 80% annexin V-positive and 63% PI-positive. IL-2-dependent CTLL-2 cells were cultured in the absence of IL-2 for 16 h to induce apoptosis, when they became PI and annexin V-positive but retained their membrane integrity.

Generation of stable transfectants

Plasmids pTriEx-3Neo-mSLPI-WS and pTriEx-3Neo-mSLPI-WN are generated by inserting the open reading frame of mouse SLPI or the coding sequence for mature SLPI without signal peptide, respectively, into an expression vector pTriEx-3Neo (Novagen, Madison, WI). Each plasmid was electroporated into RAW 264.7 cells using Gene Pulsar (Bio-Rad, Hercules, CA). Stable transfectants were selected in neomycin and cloned by limiting dilution as described previously (18, 21). A clone expressing the secretory form of SLPI (RS-3) and a clone expressing the nonsecretory form of SLPI (RS-4) were used for analyses.

Clearance of apoptotic cells by macrophages

Apoptotic cells were added to the macrophage monolayers (2 x 106 cells/10-cm plate) at a ratio of 1:20 (apoptotic CTLL-2) or 30 (apoptotic thymocytes). Zymosan A particles were opsonized with opsonizing reagents (Molecular Probes), washed, and dispersed in macrophage culture at a ratio of 1:50. Cells were incubated at 37°C for 90 min in a 3-ml final medium volume. Plates were then washed three times with FCS-free complete RPMI 1640 medium, and incubated in 2 ml of FCS-free complete RPMI 1640 medium at 37°C for 16–18 h or as specified. Where indicated, association of apoptotic cells with macrophages was evaluated. In brief, J774.1 cells in eight-well chamber slides (2.5 x 103 cells/well) were incubated with apoptotic CTLL-2 cells (5 x 104 cells/well) at 37°C for 90 min. After washing and removing of nonattached cells, slides were incubated at 37°C for an additional 16 h. J774.1 cell-associated TUNEL-positive CTLL-2 cells at the beginning and at the end of the 16-h incubation were counted after fixation of slides with 4% buffered formaldehyde followed by TUNEL labeling.

Preparation of cell lysates

Macrophages were solubilized in 20 mM Tris-HCl (pH 7.4), 1% Triton X-100, 150 mM NaCl, 2 mM EDTA on ice for 30 min. Thymuses were homogenized in extraction buffer (0.01 M Tris-HCl, pH 7.4, plus 0.15 M NaCl) following repeated freezing and thawing. Insoluble material was removed by centrifugation (25,000 x g, 15 min). Protein concentrations were determined by the Bio-Rad dye binding assay using BSA as a standard.

Immunoprecipitations

Immunoprecipitations were performed as described previously (18, 21). Cell lysates or cell supernatants were precleared by mixing with 15 µl of protein A-Sepharose for 1 h at 4°C. SLPI was immunoprecipitated by incubating the precleared lysates or the precleared supernatants with anti-mouse SLPI Ab for 3 h at 4°C. Beads were washed three times with buffer TBS (25 mM Tris, pH 7.4, 150 mM NaCl) plus 0.5% Nonidet P-40, 0.5% Triton X-100, and 1.5 mM MgCl2, followed by two washes with TBS only.

Western blot

The proteins in culture supernatants were precipitated with ice-cold 10% TCA for 30 min. The precipitates were rinsed twice in acetone containing 5 mM DTT to remove traces of TCA, and boiled for 5 min in Laemmli buffer. Alternatively, immunoprecipitates were solubilized in Laemmli buffer, separated by SDS-PAGE and transferred to a 0.1-µm pore nitrocellulose membrane (Schleicher & Schuell, Keene, NH). The membrane was blocked with 5% milk and blotted with anti-mouse SLPI IgG (1:10,000). The blots were then washed four times with PBS containing 0.02% Tween 20, and developed with a sandwich of biotinylated goat anti-rabbit IgG (ICN Pharmaceuticals, Aurora, OH) and streptavidin-HRP (Calbiochem, San Diego, CA). The immunoreactive bands were visualized with H2O2-activated diaminobenzidine.

Reverse casein gel zymography

The levels of active SLPI were analyzed by reverse casein gel zymography as described (22). For culture supernatants of macrophages, culture supernatants were concentrated 10-fold at 4°C using an Amicon Centricon 10 concentrator (Millipore, Bedford, MA) and analyzed immediately. The concentrated supernatants or the protein extracts of each thymus (50 µg) were subjected to SDS-PAGE on a 15% polyacrylamide gel containing 0.1% casein under nonreducing conditions. After electrophoresis, the gel was washed three times with 2.5% Triton X-100 for 30 min to remove SDS, rinsed with water, and incubated overnight at 37°C in chymotrypsin digestion solution containing 10 µg of chymotrypsin/ml, casein gel reaction buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM CaCl2). The gels were stained with 0.125% Coomassie brilliant blue. Protease inhibitor-protected areas appeared as blue bands. In some experiments, culture supernatants of macrophages were treated with anti-SLPI coupled-beads to remove SLPI before reverse casein gel zymography. Where indicated, densitometric analyses of gels were performed using NIH Image.

RNA analysis

Total RNA was isolated using the SV Total RNA Isolation System (Promega, Madison, WI) according to the manufacturer’s directions. Northern blots were performed as described previously (18, 21). Semiquantitative RT-PCR was performed with 1 µg of RNA using a one-step RNA PCR kit (Takara, Shiga, Japan). After a "hot-start" for 5 min at 94°C, 28–32 cycles were used for amplification, with a melting temperature of 94°C, an annealing temperature of 60°C (except IL-10 at 58°C), and an extending temperature of 72°C, each for 1 min, followed by a final extension at 72°C for 8 min. The RT-PCR products were separated on a 2% agarose gel and stained with ethidium bromide. Primers used for these analyses are: for SLPI: forward 5'-CCTCTGCTCCTGTGCACAAGT-3' and reverse 3'-TGCAGCTCAACAATAGAAATCAATT-5', amplifying a 347-bp product (18); for TNF-{alpha}: forward 5'-ACAAGCCTGTAGCCCACG-3' and reverse 3'-TCCAAAGTAGACCTGCCC-5', amplifying a 428-bp product (23); for TGF-{beta}: forward 5'-CAAGGAGGAATACAGGG-3' and reverse 3'-CGCACACAGCAGTTCTTCTC-5', amplifying a 260-bp product (24); for IL-10: forward 5'-CAACATACTGCTAACCGATC-3' and reverse 3'-TCACAGGGGAGAAATCGATG-5', amplifying a 213-bp product (25). The primers for the "housekeeping" gene {beta}-actin were forward 5'-ATGGATGACGATATCGCT-3' and reverse 3'-ATGAGGTAGTCTGTCAGGT-5', amplifying a 587-bp product (26). Densitometric analyses of gels were performed with NIH Image.

Quantitation of cytokines

Cytokine contents in macrophage-conditioned medium or in the thymus homogenates were assayed according to the manufacturer’s instruction, using ELISA kits from BioSource International (Camarillo, CA) for TNF-{alpha} and IL-10, or from R&D Systems (Minneapolis, MN) for TGF-{beta}.

Histological analysis

Five-micrometer-thick cryosections of each thymus were air-dried and fixed in acetone. For in situ detection of DNA cleavage, staining by TUNEL labeling was performed as previously described (27, 28, 29). TUNEL-positive cells were visualized with H2O2-activated diaminobenzidine. Immunohistology for detection of macrophage F4/80 was performed as previously described (28). Briefly, sections were blocked with PBS containing 5% normal goat serum, incubated with F4/80 (rat IgG2b mAb), followed by alkaline phosphate-conjugated goat ant-rat IgG Ab (Cappel, West Chester, PA). F4/80-positive staining was revealed using the Alkaline Phosphate Substrate kit (Vector Laboratories, Burlingame, CA) to color the reaction product blue. Sections were counterstained with Meyer’s hematoxylin. Slides were mounted and examined using Microflex UFX-II (Nikon, Tokyo, Japan).

Statistical analysis

Data from ELISA were analyzed by two-tailed Student’s t test, with p < 0.05 considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Induction of SLPI by exposure of macrophages to apoptotic cells in vitro

Mouse peritoneal macrophages and macrophage-like cell lines RAW264.7 and J774.1 were analyzed for their ability to produce SLPI after exposure to apoptotic cells. Either mouse thymocytes pretreated with dexamethasone or CTLL-2 cells cultured in the absence of IL-2 were used as apoptotic target cells. Macrophage monolayers were incubated for 90 min with target cells followed by extensive washing. Macrophages were then cultured in a serum-free medium for an additional 18 h at 37°C. Culture medium was collected and SLPI contents were evaluated by Western blot analysis using anti-mouse SLPI Ab (Fig. 1A). Secreted SLPI was detectable only in macrophages exposed to apoptotic target cells but not in macrophages alone. Secreted SLPI was the product of macrophages because apoptotic cells alone did not produce detectable SLPI when incubated for 24 h (Fig. 1A). Similar results were obtained in experiments using either primary macrophages or macrophage cell lines. Although RAW264.7 and J774.1 cells have different basal SLPI levels (18), both cell types secreted enhanced SLPI in response to apoptotic cells. To see whether secreted SLPI is bioactive, we tested antichymotrypsin activity in the medium of macrophages treated with apoptotic cells (Fig. 1B). In the conditioned media of both primary and RAW macrophages, a protein with a molecular mass of ~12 kDa possessed an antichymotrypsin activity (Fig. 1B). This activity was depleted from the media by immobilized anti-SLPI, but not by control IgG-conjugated beads. These results show that macrophages produce bioactive SLPI upon treatment with apoptotic cells. To test whether phagocytosis per se triggers SLPI production, primary macrophages and RAW264.7 were exposed to zymosan particles opsonized with Ig. Although these cells efficiently engulfed opsonized zymosan, phagocytosis of these particles failed to produce SLPI as assessed by reverse casein gel zymography (Fig. 1B) and Western blots (not shown).



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  		FIGURE 1. Murine macrophages produce SLPI during clearance of apoptotic cells. Monolayers of primary macrophages of BALB/C mice, J774.1 cells, or RAW264.7 cells were incubated alone (lane 1), with early (lane 2) or late (lane 3) apoptotic thymocytes, apoptotic CTLL-2 cells (lane 4), Ig-opsonized zymosan (lane 5) for 90 min. After removal of target cells or particles, macrophages were incubated in FCS-free RPMI 1640 medium for an additional 18 h. As controls, macrophages were incubated in the presence of LPS (100 ng/ml) for 18 h (lane 6), or apoptotic cells were incubated alone for 18 h. The levels of SLPI expression (A) and active SLPI (B) in the culture medium were assessed by immunoblot and reverse zymography analysis, respectively. Lane 7, the culture supernatants from lane 3 were cleared with anti-SLPI-coupled Sepharose; lane 8, the culture supernatants from lane 3 were cleared with normal rabbit IgG-coupled Sepharose. Results are representative of five similar experiments.

 
We next analyzed the kinetics of SLPI expression and secretion in primary mouse macrophages exposed to apoptotic cells. Monolayers of primary macrophages were cultured with or without early apoptotic thymocytes for 90 min at 37°C. After removal of noningested apoptotic cells, the cells were cultured at 37°C for an additional 0, 3, 6, 9, or 15 h. At mRNA levels, an increase of SLPI by apoptotic cell-treated macrophages was evident at 6 h and remained elevated (Fig. 2A). SLPI transcripts in the apoptotic thymocytes were undetectable by RT-PCR (not shown). Secreted SLPI in culture medium was detectable after 9 h (Fig. 2B).



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  		FIGURE 2. Kinetics of SLPI expression in macrophages phagocytosing apoptotic cells. Adherent primary macrophages were pretreated with or without early apoptotic thymocytes as in Fig. 1, and incubated alone for additional times as indicated. A, Semiquantitative RT-PCR was performed using total RNA as described in Materials and Methods; lower panel, equal loading as defined by the levels of {beta}-actin mRNA. B, The levels of SLPI in the culture medium were analyzed by Western blots. These results are representative of three similar experiments.

 
SLPI expression in thymuses after administration of anti-CD3 Ab in vivo

SLPI is reported to be present in murine thymus (30). Preferential apoptosis of the CD4+CD8+ thymocyte subset by anti-CD3 Ab has been well-documented in vivo as well as in vitro (31, 32, 33, 34). Previous studies indicate that thymic macrophages are the principal stromal cells responsible for phagocytosis of dying thymocytes and that the number of TUNEL-positive thymocytes declines to the original basal level after apoptotic cells are phagocytosed by macrophages in the thymus following administration of anti-CD3 Ab or hydrocortisone (28, 35). To examine whether SLPI might be up-regulated during macrophage phagocytosis of apoptotic thymocytes in vivo, we injected 50 µg of anti-CD3 Ab into BALB/c mice i.p. At different intervals, thymuses were removed and sectioned for staining of apoptotic cells and macrophages (Fig. 3A). TUNEL staining analysis showed that an increase in apoptotic cells was detectable in the cortical region of the thymus as early as 13 h after injection (Fig. 3Ab). A massive increase in apoptotic cells was evident within 16 h, and a significant number of TUNEL-positive thymocytes were scattered throughout the cortical area of the thymus (Fig. 3Ac). Double staining showed that most apoptotic cells in the cortex were situated with F4/80+ macrophages (Fig. 3Ad). The number of TUNEL-positive thymocytes decreased rapidly by 20 h (Fig. 3A, compare c and e). By 24 h, TUNEL-positive thymocytes were reduced drastically in number and apoptotic cells were restricted to the cortex of thymus (Fig. 3Ag), whereas F4/80+ macrophages were found throughout the thymus (Fig. 3Ah).



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  		FIGURE 3. Detection of apoptotic cells, macrophages, and SLPI in murine thymus after administration of anti-CD3 Ab. A, Seven-week-old BALB/c mice were injected i.p. with 50 µg of anti-CD3 Ab (2C11-145) and each thymus was removed at 0 (a), 13(b), 16 (c and d), 20 (e and f), or 24 h (g and h) postinjection. Cryostat sections of thymus were stained by TUNEL method (b, c, e, and g) or with F4/80 Ab (h), or with both (a, d, and f), and counterstained with Meyer’s hematoxylin. Thymic cortex (C) and medulla (M) are indicated; brown (arrows), TUNEL+ cells; blue (arrowheads), F4/80+ macrophages. Magnifications are x40 except for d and f (x250). These sections are representative of four mice for each condition. B, Total RNA was isolated from each thymus, and semiquantitative RT-PCR was performed for SLPI and {beta}-actin. Fold of SLPI induction is defined as the ratio of relative signal (SLPI/actin) in treated vs 0-h sample. Results are shown from a representative of three independent experiments. C, Thymus extracts were prepared as described in Materials and Methods. Active SLPI contents were determined by reverse zymography analysis. Fold of induction was quantified by dividing the intensity of SLPI signals to that of 0-h sample. Results are representative of three independent experiments.

 
SLPI expression in the thymus after administration of anti-CD3 Ab was evaluated at both the mRNA and the protein level. Total RNA was isolated from each thymus after the administration of anti-CD3 Ab for different durations, and SLPI transcripts were assessed by semiquantitative RT-PCR analysis. SLPI expression was increased and reached the peak by 16 h after administration of anti-CD3 Ab (Fig. 3B). The levels of active SLPI in the thymus were assessed by reverse zymography analysis, and found to be increased 16–20 h after administration of an anti-CD3 Ab (Fig. 3C). It remains to be determined whether SLPI was only produced in those macrophages that had taken up apoptotic cells.

Expression of cytokines in thymuses after administration of anti-CD3 Ab

To examine the expression of other pro- or anti-inflammatory cytokines in the thymus after administration of anti-CD3 Ab, semiquantitative RT-PCR was performed (Fig. 4A). TNF-{alpha} mRNA was increased 10 h after the injection. TGF-{beta} mRNA expressed abundantly in the thymus of control mice and was increased within 13 h in response to anti-CD3 injection, and remained elevated up to 24 h. IL-10 transcription was up-regulated within 16 h of the injection, increasing up to 20–24 h. At the protein level, the concentration of these three cytokines also showed elevations, and all cytokine levels reached a peak at 20 h after administration of anti-CD3 Ab (Fig. 4B). The levels of active TGF-{beta} were only ~15% of total TGF-{beta} (data not shown). These results suggest that clearance of apoptotic cells by macrophages is accompanied by a change in cytokine networks, which may serve to control the extent of the inflammation reaction during the clearance.



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  		FIGURE 4. Cytokine profile in murine thymus after administration of anti-CD3 Ab. Thymuses from anti-CD3-injected mice were prepared as in Fig. 3. A, Total RNA was isolated from each thymus, and semiquantitative RT-PCR was performed for TNF-{alpha}, TGF-{beta}, and IL-10. The lower bands demonstrate equal loading as defined by content of {beta}-actin mRNA. Results are representative of three independent experiments. B, The cytokine levels of the extract of each thymus were determined by ELISA. Results are shown as mean ± SD from four mice, and are representative of three independent experiments. *, p < 0.05, and **, p < 0.01 compared with 0-h group.

 
Colchicine inhibits uptake of apoptotic cells but not SLPI production by macrophages

To determine whether the enhanced SLPI production by macrophages was caused by recognition or engulfment of apoptotic cells, J774.1 cells were preincubated with colchicine (1 or 10 µM) for 1 h before addition of apoptotic CTLL-2 cells. After 90 min of coculture, unbound apoptotic cells were removed. J774.1 cell associated TUNEL-positive cells were counted under the microscope. Colchicine at both concentrations did not block the association of J774.1 cells with apoptotic CTLL-2 (Fig. 5A). Thereafter, macrophages were cultured in the presence or absence of colchicine for an additional 16 h. The number of J774.1 surface-associated TUNEL-positive cells was decreased drastically in control J774.1. In contrast, when cultured in the presence of colchicine, most of the apoptotic cells remained on the cell surface of J774.1 (Fig. 5A). However, treatment of colchicine had no effect on SLPI induction (Fig. 5B). Similar results were obtained in experiments using RAW264.7 cells or murine primary macrophages (not shown). Thus, engulfment of apoptotic cells is not necessary for SLPI induction, suggesting that recognition of apoptotic cells by macrophage surface receptors may be sufficient in inducing SLPI.



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  		FIGURE 5. Colchicine inhibits engulfment of apoptotic cells without affecting SLPI production in macrophages. J774.1 cells were pretreated with indicated concentrations of colchicine for 1 h before incubation with apoptotic CTLL-2 for 90 min. After removal of unbound cells, macrophages were incubated with the same concentrations of colchicine for an additional 16 h. A, Numbers of J774.1-associated TUNEL-positive CTLL-2 before ({square}) or after ({blacksquare}) 16-h incubation were scored as described in Materials and Methods. Results are expressed as the number of apoptotic cells per macrophage and are means ± SE from three experiments. B, SLPI levels in the culture supernatants were determined by Western blot analysis. The result is representative of three independent experiments.

 
IFN-{gamma} inhibits SLPI, but enhances TNF-{alpha}- production in macrophages exposed to apoptotic cells

We previously showed that IFN-{gamma} inhibited both basal and LPS-inducible SLPI expression in macrophages (18). To test whether IFN-{gamma} also inhibits macrophage SLPI production in response to apoptotic cells, primary macrophages were incubated with IFN-{gamma} for 6 h. SLPI mRNA expression was inhibited with IFN-{gamma}, as assessed by RT-PCR (Fig. 6A). These macrophages were incubated with or without apoptotic CTLL-2 for 90 min. After removing unbound target cells, macrophages were then cultured for an additional 16 h at 37°C in the presence of the same concentration of IFN-{gamma} under serum-free conditions. Secreted SLPI in the culture medium was estimated by Western blot analysis (Fig. 6B). IFN-{gamma} at a concentration of 100 U/ml completely inhibited SLPI production. The levels of TNF-{alpha} in the culture supernatants of macrophages were examined by ELISA (Fig. 6C). Macrophages did not produce TNF-{alpha} in the presence of IFN-{gamma} alone. Target CTLL-2 cells themselves produced no detectable levels of TNF-{alpha} (<19.5 pg/ml), even when incubated for 24 h (data not shown). Exposure of macrophage monolayers to apoptotic CTLL-2 resulted in TNF-{alpha} secretion, which was increased by IFN-{gamma} treatment. There appears to be an inverse correlation between SLPI and TNF-{alpha} levels in response to apoptotic cells and IFN-{gamma}.



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  		FIGURE 6. Effects of IFN-{gamma} on SLPI expression and TNF-{alpha} production in macrophages exposed to apoptotic cells. Primary mouse macrophages were pretreated with indicated concentrations of IFN-{gamma} for 6 h before incubation with apoptotic CTLL-2 for 90 min. After removal of unbound target cells, macrophages were incubated with the same concentrations of IFN-{gamma} for an additional 16 h. A, SLPI expression was determined after pretreatment with IFN-{gamma}, as assessed by semiquantitative RT-PCR. The lower bands demonstrate equal loading as defined by content of {beta}-actin mRNA. B, SLPI levels in the culture supernatants were determined after the additional 16-h cultivation, by Western blot analysis. C, The levels of secreted TNF-{alpha} were quantified after the additional 16-h cultivation, by ELISA. These results are representative of four independent experiments. **, p < 0.01, compared with macrophages without IFN-{gamma}.

 
SLPI overexpression leads to the suppression of TNF-{alpha} production in response to apoptotic cells

We have previously reported that transfection of SLPI suppressed macrophage production in response to LPS (18, 21). To determine whether SLPI expression has any impact on apoptotic cell-induced TNF-{alpha} production in macrophage, we generated two stable SLPI cDNA transfectants from RAW264.7 cells. RS-3 overexpresses a secretory form of SLPI, whereas RS-4 overexpresses a nonsecretory form of SLPI. By Northern blot analysis, SLPI mRNA was easily detectable in RS-3 and RS-4 (Fig. 7A). The slower electrophoretic mobility of SLPI mRNA in RS-3 and RS-4 compared with the endogenous SLPI transcripts in RAW264.7 is attributable to an extra 2.3-kb sequence acquired from the vector. Intracellular retention of SLPI protein in these transfectants was confirmed by Western blotting. RS-3 expressed a robust level of SLPI, which can be detected both in the supernatant and the cell lysate, while SLPI expressed in RS-4 was detected only in the cell lysate. Parental RAW264.7 expressed little SLPI (Fig. 7A). To test the effect of SLPI expression on TNF-{alpha} production in response to apoptotic cells, RAW264.7, RS-3 and RS-4 cells were incubated with apoptotic CTLL-2 for 90 min, removed, and then incubated for an additional 18 h. TNF-{alpha} levels in the medium were assessed by ELISA. TNF-{alpha} production in both transfectants was greatly reduced, compared with that in parental RAW264.7 cells (Fig. 7B), even though both transfectants exhibited similar binding and engulfing activity toward target cells (data not shown). These cells also had a much reduced sensitivity to LPS as judged by LPS-induced TNF-{alpha} (Fig. 7C), consistent with our previous reports using other SLPI transfectants (18, 21).



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  		FIGURE 7. SLPI overexpression in RAW264.7 cells leads to a suppression of TNF-{alpha} production in response to apoptotic cells. A, Characterization of stable cells RS-3 and RS-4. Northern blot and Western blot analyses for SLPI were performed on parental RAW264.7 cells, SLPI transfectants RS-3 and RS-4. The Northern blot (20 µg of RNA) was probed with the mouse SLPI open reading frame, and the same membrane was reprobed with an actin oligonucleotide as a loading control. SLPI was immunoprecipitated from conditioned media (CM) or cell lysates as indicated with rabbit anti-SLPI IgG and blotted with the same Ab. B, Suppression of TNF-{alpha} production in response to apoptotic cells in SLPI transfectants. Cells at a density of 5 x 105/ml were incubated either alone or with apoptotic CTLL-2 cells at a ratio of 1:20 (macrophage-apoptotic cells) for 90 min. Unbound target cells were removed, and the cells were then cultured for an additional 18 h. C, Suppression of LPS-induced TNF-{alpha} production in SLPI transfectants. Cells at a density of 5 x 105/ml were treated with indicated concentrations of LPS. The conditioned media were collected 24 h later for TNF-{alpha} analysis. Concentrations of TNF-{alpha} in culture media were assessed by ELISA (B and C). Results are means ± SD of triplicates from one of four similar experiments. Some error bars fall within the symbols.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This is the first report that murine macrophages synthesize and secrete SLPI during clearance of apoptotic cells (Figs. 1 and 2). This phenomenon was detected in experiments with primary macrophages from the peritoneal cavity of BALB/c mice as well as macrophage cell lines RAW264.7 and J774.1, where apoptotic thymocytes or CTLL-2 cells were used as target cells. Increased SLPI expression was also observed in the thymuses of BALB/c mice that had been injected with anti-CD3 Ab to induce thymocyte apoptosis (Fig. 3). Colocalization of apoptotic thymocytes with macrophages (Fig. 3A) suggests that thymic macrophages were the likely source of SLPI induced upon phagocytosis of apoptotic thymocytes in vivo. SLPI is present in the thymus of normal mice (30). Because the majority of thymocytes die, clearance of dying thymocytes by resident thymic macrophages is the most plausible explanation for the presence of SLPI in the thymus of normal mice.

SLPI production was undetectable upon phagocytosis of Ig-opsonized zymosan by macrophages, indicating that phagocytosis per se is not sufficient to induce SLPI secretion. Colchicine was shown to inhibit the internalization of apoptotic cells by macrophages, but did not affect the interaction of apoptotic cells with macrophage surface receptors (36). In this work, the treatment of macrophages with colchicine blocked the uptake of apoptotic cells but did not affect SLPI induction, suggesting that SLPI is produced through the interaction of apoptotic cells with macrophage surface receptors. A number of molecules have been postulated to participate in the recognition of apoptotic cells by macrophages. These include phosphatidylserine receptor (37), vitronectin receptors (38, 39), an LPS-binding molecule CD14 (40), and integrins (37, 41). It remains to be determined which macrophage surface receptor(s) is/are involved in the induction of SLPI expression in response to apoptotic cells.

Two apoptotic target models were used in the present study: dexamethasone-treated thymocytes and IL-2-depleted CTLL-2 cells. Both targets are able to induce SLPI from macrophages. In most experiments, these two sources are interchangeable except in TNF-{alpha} induction experiments. CTLL-2 cells induced primary macrophages J774.1 or RAW264.7 to secrete substantial levels of TNF-{alpha}, whereas apoptotic thymocytes induced little but detectable TNF-{alpha} (not shown). Fadok and coworkers (42) suggested that a contamination of necrotic populations in CTLL-2 cells might explain why uptake of these cells could be proinflammatory. In contrast, Kurosaka et al. (43) and Uchimura et al. (44) have used CTLL-2 cells in their studies and carefully examined these cells for apoptotic markers and membrane integrity. They found that 12 h after withdrawal of IL-2 from the cultures, CTLL-2 cells became PI and annexin V-positive with DNA ladder formation, typical of apoptotic cells. No membrane leakage was found up to 24 (43) or 28 h (44) after removal of IL-2, as assayed by the release of lactate dehydrogenase. We also found that the majority of CTLL-2 cells appeared to die via apoptosis and believe that it was apoptotic CTLL-2 cells that triggered macrophages to produce proinflammatory cytokine TNF-{alpha}. It is not known at present why apoptotic CTLL-2 and apoptotic thymocytes have different potencies in inducing TNF-{alpha} from macrophages. A recent report by Perskvist et al. (45) showed that phagocytosis of apoptotic neutrophils markedly increased TNF-{alpha} production by human macrophages. Thus, apoptotic cells with different origins may differ in their ability to induce inflammatory cytokines during their clearance by macrophages, perhaps due to their distinct surface presentations.

We have previously reported that IFN-{gamma} suppresses SLPI expression and restores LPS responsiveness in SLPI-producing cells (18). In this study, we showed that IFN-{gamma} inhibited the basal expression of SLPI in primary mouse macrophages as well as the inducible SLPI in response to apoptotic cells (Fig. 6). However, pretreatment of macrophages with IFN-{gamma} augmented TNF-{alpha} production under the same conditions (Fig. 6). The inverse correlation between TNF-{alpha} and SLPI is suggestive of the role of SLPI as an anti-inflammatory mediator during phagocytosis of apoptotic cells. To address this issue, we compared TNF-{alpha} production by SLPI transfectants in response to apoptotic cells with their parental RAW264.7 cells. SLPI overexpression resulted in the suppression of TNF-{alpha} production (Fig. 7). Given the slow kinetics of SLPI expression in macrophages exposed to apoptotic cells, it is unlikely that inducible SLPI inhibits TNF-{alpha} production in the same macrophage population in an autocrine fashion. Rather, SLPI produced by existing macrophages after contact with apoptotic cells may serve to prevent overproduction of TNF-{alpha} during phagocytosis of apoptotic cells by incoming macrophages.

Balance between pro- and anti-inflammatory mediators is critical in maintaining an immune homeostasis during phagocytosis of apoptotic cells. Recent studies indicate that TGF-{beta} produced by apoptotic cells as well as phagocytes have a major role in the anti-inflammatory effect during clearance of apoptotic cells (3, 4, 5, 46). Uptake of apoptotic cells by macrophages facilitated the growth of trypanosomes in Chagas’ disease in a TGF-{beta} and PGE2-dependent manner, thereby promoting disease progression (47). Although J774.1 cells were also found to produce TGF-{beta} following exposure to apoptotic cells (3, 4, 5), we could not detect TGF-{beta} production in J774.1 cells in response to apoptotic CTLL-2 cells (data not shown). However, we did detect an increase in the TGF-{beta} level in mouse thymuses in response to apoptosis of thymic T cells (Fig. 4). We also observed an increase in the TNF-{alpha} and IL-10 levels in the thymuses after administration of anti-CD3 Ab. Thus, the increased expression of TNF-{alpha}, TGF-{beta}, and IL-10 may serve as regulatory mediators for inflammatory responses in the thymus after administration of anti-CD3 Ab. Two recent studies indicated that noninflammatory clearance of apoptotic cells could be regulated by anti-inflammatory lipid mediators (48, 49). Our findings suggest SLPI can also participate in the resolution of inflammatory responses.

Resolution of the inflammatory response in acute cutaneous wounds is critical to healing, and delays in this process often lead to impaired wound maturation and closure. Apoptotic cells are identified in the wounding site by histological examination of tissue sections and by in situ DNA fragment labeling (50), which may contribute to an augmented inflammatory response in the absence of anti-inflammatory mediators such as TGF-{beta} and SLPI. Delayed wound healing has been observed in TGF-{beta}1-null mice (51). Recently Ashcroft et al. (52) reported that SLPI-null mice had impaired cutaneous wound healing with increased inflammation. Our results imply that SLPI plays a role in controlling the inflammatory response in the acute cutaneous wound during phagocytosis of apoptotic cells, and may help to explain why wound healing is impaired in SLPI-null mice (52).


   Footnotes
 
1 This work was in part supported by National Institutes of Health Grants GM61710/AI30165 (to A.D.). The Department of Microbiology & Immunology of Weill Medical College acknowledges the support of the William Randolph Hearst Foundation. Back

2 Address correspondence and reprint requests to Dr. Chikako Odaka, Department of Safety Research on Blood and Biological Products, National Institute of Infectious Diseases, 1-23-1, Toyama, Shinjuku-ku, Tokyo, 162-8640, Japan. E-mail address: odaka{at}nih.go.jp Back

3 Abbreviations used in this paper: SLPI, secretory leukocyte protease inhibitor; PI, propidium iodide. Back

Received for publication November 15, 2002. Accepted for publication May 21, 2003.


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 Discussion
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