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J Appl Physiol 104: 601-609, 2008. First published November 29, 2007; doi:10.1152/japplphysiol.00254.2007
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Apoptotic myocytes generate monocyte chemoattractant protein-1 and mediate macrophage recruitment

Miyuki Kobara,1 Nahoko Sunagawa,1 Masaki Abe,1 Nana Tanaka,1 Hiroe Toba,1 Hironori Hayashi,2 Natsuya Keira,2 Tetsuya Tatsumi,2 Hiroaki Matsubara,2 and Tetsuo Nakata1

1Department of Clinical Pharmacology, Kyoto Pharmaceutical University; and 2Department of Cardiovascular Medicine, Kyoto Prefectural University of Medicine, Kyoto, Japan

Submitted 4 March 2007 ; accepted in final form 27 November 2007


   ABSTRACT
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 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
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The mechanisms by which apoptotic myocytes are removed by macrophages have not been fully elucidated. This study examined whether apoptotic myocytes actively recruit macrophages by generating monocyte chemoattractant protein-1 (MCP-1) in experiments in vitro and in vivo. Neonatal rat cardiac myocytes were incubated for 4 h in the presence or absence of staurosporine (STS, 0.2–1 µmol/l), an apoptosis inducer. Nuclear staining with DAPI showed that STS induced apoptosis in a dose-dependent fashion. STS (1 µmol/l) caused extensive DNA fragmentation and increased caspase-3 activity compared with a serum-deprived control. MCP-1 mRNA and protein levels in myocytes increased twofold and fourfold, respectively, on STS treatment, and immunochemical staining revealed that apoptotic myocytes expressed MCP-1. To elucidate the role of MCP-1 expressed in apoptotic myocytes to recruit macrophages/monocytes, rat monocytes were incubated in the supernatant of STS-treated myocytes using a trans-well system. The culture medium of STS-treated myocytes recruited monocytes in a MCP-1-dependent fashion. In addition, experiments were performed in vivo using ischemia-reperfused rat hearts. Rats were subjected to 30 min of ligation of the left coronary artery followed by 24 h of reperfusion. After the reperfusion, in the ischemic border myocardium, 17.1 ± 1.1% of myocytes were terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) positive. Moreover, double staining using the TUNEL technique and immunohistochemistry with MCP-1 antibody showed that 69.8 ± 3.9% of TUNEL-positive myocytes expressed MCP-1 protein. Concomitantly, activated macrophages infiltrated the areas of apoptosis remarkably. These results suggest that apoptotic myocytes produce MCP-1, which have a critical role in the active recruitment of macrophages.

apoptosis; cardiac myocytes

NECROSIS AND APOPTOSIS are regarded as two distinct forms of lethal cell injury. Apoptosis of cardiac myocytes has been reported in several cardiovascular diseases, such as end-stage heart failure (20, 21), myocardial infarction (10, 25, 39), dilated cardiomyopathy (45), and arrhythmogenic right ventricular cardiomyopathy (15, 38). In addition, the apoptosis of myocytes independently contributes to cell loss from necrosis and plays a role in cardiac dysfunction (12).

Necrosis is a passive process, resulting in a disruption of the cellular membrane and remarkable inflammation. On the other hand, apoptosis is achieved by a tightly controlled death program, and the rapid removal by phagocytes is supposed to ensure a quiet clearance of apoptotic cells without remarkable inflammation (22). There is evidence to suggest that the impaired clearance of apoptotic cells contributes to the pathogenesis of certain diseases (2, 6). With regard to the process by which apoptotic cells are removed, several reports are available on the phagocytic recognition of apoptotic cells. Four types of molecules on apoptotic cells have been proposed to be involved in the targets of phagocytosis by macrophages: asialoglycoprotein (4), thrombospondin 1 (26), phosphatidylserine (5, 14), and iC3b (34). However, little is known about whether apoptotic cells are active participants in the recruitment of phagocytes.

CXC chemokines, represented by IL-8, exhibit strong chemotactic activity for neutrophils, while CC chemokines are implicated in the activation of macrophages/monocytes and lymphocytes (46). Among the CC chemokines, monocyte chemoattractant protein-1 (MCP-1) recruits monocytes from the general circulation to inflammatory sites (19). Therefore, we examined the hypothesis that apoptotic myocytes produce MCP-1, which has a critical role in the recruitment of monocytes/macrophages.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
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Chemicals

Staurosporine (STS) was generously provided by Kyowa Medex (Tokyo, Japan). All animals were handled in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH publication No. 85-23, Revised 1996). The protocol was approved by the Bioethics Committee of Kyoto Pharmaceutical University.

In Vitro Experiments

Culture of neonatal rat cardiac myocytes.   Primary cultures of rat neonatal cardiac myocytes were prepared as previously described (16). Briefly, 1- or 2-day-old Wistar rats were anesthetized with ether, and the hearts were removed and placed in PBS. The atria were discarded, and ventricles were minced and dissociated with 0.2% type I collagenase (Sigma, St. Louis, MO). Cell suspensions were centrifuged at 300 g for 5 min and resuspended in HEPES buffer (pH 7.35) containing (in mmol/l) 116 NaCl, 5.1 KCl, 0.8 MgSO4, 1.0 NaH2PO4, 20 HEPES, and 5.5 glucose. Then the cells were layered onto a Percoll density gradient (density 1.059/1.082 g/ml) and centrifuged at 1,000 g for 30 min (30). The myocyte layer at the Percoll interface was carefully collected and washed in HEPES buffer. The myocytes were resuspended and seeded into 60-mm culture dishes (1 x 105 cells/cm2). The myocytes were incubated in DMEM (Nissui Pharmaceutical) supplemented with 10% FBS and antibiotics (5,000 µg/ml gentamicin, 5,000 µg/ml ampicillin, and 100 µg/ml amphotericin B). Bromodeoxyuridine (BrdU, 100 µmol/l) was added during the first 48 h to prevent the proliferation of nonmyocytes. The myocytes were then incubated in DMEM containing 0.5% FBS without BrdU, and all experiments were done 36–48 h after this incubation. By using this approach, we routinely obtained contractile cultures with >95% myocytes, as assayed by immunochemical staining for myosin heavy chain.

Experimental protocol.   Cultured myocytes were washed twice with PBS, followed by a final incubation in serum-deprived medium. During this final incubation, cardiac myocytes were treated with or without STS (0.2–1 µmol/l), an apoptosis inducer. Unless indicated otherwise, treatment was continued for 4 h, at which time we examined the apoptosis of myocytes, caspase-3 activity, MCP-1 production, IL-1β production, and the migration of monocytes.

Histochemical determination of apoptosis.   Myocytes were grown on type I collagen-coated glass cover slips. After the administration of STS for 4 h, cultured cells were fixed with 2% buffered formalin for 5 min and permeabilized with 0.5% Triton X-100 for 5 min. To identify myocytes, cells were treated with 10% normal goat serum and incubated with the mouse monoclonal anti-rat myosin heavy chain antibody (1:50, Medao) for 1 h. After being washed with PBS, cells were incubated with fluorescein-conjugated anti-mouse IgG for 1 h. Then nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI). An average of 800–1,000 myocytes from random fields were analyzed for each experiment, and counts of apoptotic myocytes were expressed as a percentage of the total nuclei counted.

Caspase-3 activity.   Caspase-3 activity was determined using an APOPCYTO Caspase-3 Colorimetric Assay Kit (MBL, Nagoya, Japan), which detects the production of the chromophore p-nitroanilide after its cleavage from the peptide substrate DEVD-p-nitroanilide (p-NA) as previously described (32). According to the manufacturer's directions, cultured myocytes were solubilized and aliquots of lysates were reacted with DEVD-p-NA for 4 h at 37°C. Activity was quantified by measuring absorbance at 405 nm using a microplate reader.

DNA extraction and gel electrophoresis.   DNA ladder formation was assayed by gel electrophoresis of low-molecular-weight genomic DNA from myocytes. Briefly, myocytes were collected by scraping and centrifuged at 1,000 g for 5 min. Pellets were then incubated with lysis buffer (10 mmol/l EDTA, 10 mmol/l Tris·HCl, pH 7.4, and 0.5% Triton X-100), and centrifuged at 15,000 g for 10 min. The supernatant was treated with RNase A (400 µg/ml) for 1 h at 37°C and then treated with proteinase K (400 µg/ml) for 1 h at 37°C. Isopropanol (final concentration, 50%) and NaCl (final concentration, 500 mmol/l) were added, and the mixture was stored overnight at –30°C. An aliquot of the mixture was centrifuged at 16,000 g for 15 min to collect DNA. After the supernatant was removed, 20 µl of TE buffer (10 mmol/l Tris·HCl, pH 7.4, and 1 mmol/l EDTA, pH 8.0) was added to dissolve the collected DNA. Ten microliters of extracted DNA was subjected to electrophoresis on a 2% agarose gel. The electrophoresis was conducted at 50 V in a flatbed gel apparatus (Mupid; Advance, Tokyo, Japan). The agarose gels were stained with 0.1% SYBR Green, and DNA was visualized with a UV transilluminator.

RT-PCR for MCP-1 mRNA.   The determination of MCP-1 mRNA was performed by reverse transcription (RT)-PCR. Myocytes were washed with ice-cold PBS twice. Total RNA was isolated using Isogen (Wako, Japan) as suggested by the manufacturer. The concentration of RNA was determined using a GeneQuant (Bio-Rad). For RT-PCR (Super script II), 1 µg of RNA was used according to the manufacturer's instructions. Each PCR had the following profile: 94°C for 1 min (1 cycle); and 94°C for 1 min, 56°C (MCP-1) or 57°C (GAPDH) for 1 min; and 72°C for 2 min with a variable number of cycles and a final extension phase at 72°C for 5 min. The number of cycles was 26 for MCP-1 and 24 for GAPDH. The primers used for the analysis of mRNA were as follows: MCP-1 forward, 5'-tgt tca gca ttg ctg cct gt-3'; MCP-1 reverse, 5'-gat ctc act tgg ttc tgg tc-3'; GAPDH forward, 5'- tcc ctc aag att gtc agc aa-3'; GAPDH reverse, 5'-aga tcc aca acg gat aca tt-3'. PCR products were run on a 2% agarose gel containing 0.5 µg/ml ethidium bromide.

Western blot analysis of MCP-1.   The amount of MCP-1 protein was determined by Western blotting. Myocytes were scraped and pelleted and incubated in 50 µl of lysis buffer [2x PBS, 1% NP-40, 0.5% deoxycholic acid, 0.1% sodium dodecyl sulfate, 1 mmol/l PMSF, and 1% protease inhibitor cocktail (Nacarai Tesuque, Kyoto, Japan); pH 7.5] for 10 min at 4°C. The suspension was centrifuged at 14,000 g for 10 min at 4°C, and the supernatant was collected as a protein extract. Immunoblotting was performed using standard protocols. Samples containing equal amounts of protein were subjected to electrophoresis on 12% SDS-polyacrylamide gels and blotted onto polyvinylidine difluoride membranes (ATTO, AE-6665). After blocking with 5% nonfat dry milk, the membranes were incubated with an antibody specific to MCP-1 (1:30 dilution, IBL) at 4°C overnight, followed by horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (Amersham, Little Chalfont, UK) at room temperature for 2 h. Finally, ECL detection reagents (Amersham, UK) were employed to visualize the peroxidase reaction product.

Immunochemical detection of MCP-1.   After treatment with STS (1 µmol/L) for 4 h, cultured cells were fixed and permeabilized. Myocytes were blocked with 10% normal goat serum and incubated with polyclonal anti-rat MCP-1 (1:20 dilution, IBL) at 4°C overnight. The cells were then incubated with a 1:100 dilution of fluorescein-conjugated goat anti-rabbit IgG for 1 h. To identify myocytes, cells were incubated with the mouse monoclonal anti-rat myosin heavy chain antibody and visualized with rhodamin-conjugated anti-mouse IgG. Then, finally, nuclei were stained with DAPI.

Microchemotaxis assay.   Chemotactic effects of culture supernatants from apoptotic myocytes were studied using a trans-well chamber (Corning). Rat peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation (Ficoll, Pharmacia, Piscataway, NJ), and monocytes/macrophages were isolated from PBMCs by adherence to culture dishes (11, 24). Briefly, autologous platelet-free plasma was incubated on 2% gelatin-coated dishes at 37°C for 1 h. Then plasma was gently washed with RPMI containing 2% FBS. PBMCs were suspended in RPMI containing 10% FBS and then incubated for 1 h at 37°C in the coating dish. Nonadherent cells were removed, and adherent cells were deadhered with EDTA (5 mmol/l)-containing RPMI. More than 80% of the adherent cells (monocytes) expressed CD172a antigen as determined by a FACS analysis. Then the adherent cells were collected and resuspended in DMEM containing 10% FBS at a final concentration of 2.8 x 105 cells/ml. The culture mediums harvested from 1) control myocytes and 2) apoptotic myocytes (4 h after STS treatment), as well as 3) fresh medium with STS and 4) fresh medium without STS, were passed through a 0.22-µm-membrane filter and placed in the bottom chamber (500 µl). A monocyte suspension (350 µl) was then placed in the trans-well insert. After 3 h of incubation, the cells that had migrated to the bottom chamber were collected, and trypan blue-extracting viable cells were enumerated with a hemacytometer. In separate experiments, fifth and sixth media were created by addition of rabbit anti-rat MCP-1 neutralizing antibody (0.03 µg/ml, PeproTec EC) and nonspecific rabbit IgG (0.03 µg/ml, DAKO), respectively, from apoptotic myocytes (see 2 above) and placed in the bottom plate 30 min before the trans-well inserts.

ELISA for IL-1β.   Protein extracts from myocytes were prepared as for the assay of MCP-1. The amount of IL-1β protein was determined by ELISA with a commercially available kit (Techene).

In Vivo Experiments

Surgical procedure.   Male Sprague-Dawley rats (250–350 g) were anesthetized with an intraperitoneal injection of pentobarbital sodium (30 mg/kg body wt). After endotracheal intubation, controlled ventilation with room air was performed using a rodent respirator (Shinano). After thoracotomy at the fourth intercostal space, the heart was exteriorized, and a 6-0 silk ligature with a needle was passed under the left coronary artery. Both ends of the ligature were passed through the polyethylene tube (PE-50) to occlude the coronary artery. The heart was subjected to 30 min of coronary artery occlusion followed by 24 h of reperfusion. Cyanosis of the anterior left ventricle demonstrated a successful coronary occlusion.

In situ detection of nuclear DNA fragmentation.   The apoptotic cell death was evaluated by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) method using an APOP TAQ kit (Chemicon) as previously described (13). After reperfusion, the heart was quickly removed and washed in ice-cold PBS and incubated in 4% buffered formalin overnight at 4°C. Sections were cut to a thickness of 4 µm and mounted on aminopropylsilane-coated glass slides. In these myocardial infarction models, interventricular septum is nonischemic. The infarcted area was defined as the necrotic region from the morphological appearance of the tissues with hematoxylin-eosin staining, and the ischemic border area was defined as a 1-mm region bordering the necrotic region (46a). Another sequential section of the heart was deparaffinized with xylene and washed with ethanol. The sections were incubated with proteinase K (20 µg/ml) for 15 min at room temperature and treated with 3% hydrogen peroxide to block the internal peroxidase activity. After the equilibrium buffer was applied to the specimens, the tissues were incubated with terminal deoxynucleotidal transferase (TdT) at 37°C for 1 h. The slides were moved to a coplin jar filled with stop solution (supplied in the kit) and incubated for 10 min at room temperature. After a wash in PBS, the anti-digoxigenin-peroxidase was applied to the specimens and incubated for 30 min at room temperature. Then the slides were placed in a 3,3'-diaminobenzidine solution for 5 min at room temperature. The sections were counterstained with hematoxylin to visualize the nuclei. Nuclei located in myocytes were recognized as myocyte nuclei. We counted 1,000 myocyte nuclei in the ischemic border region using a light microscope and calculated the proportion of TUNEL-positive nuclei.

DNA extraction and gel electrophoresis.   After reperfusion, low molecular DNA was isolated from cardiac tissue by standard techniques. In brief, freshly isolated cardiac tissues of the ischemic border segments (20 mg) were minced into small pieces and homogenized in 300 µl of lysis buffer (10 mmol/l Tris·HCl, pH 7.4, 10 mmol/l EDTA, and 0.5% Triton X-100) using a polytron homogenizer. Aliquots of the homogenate were incubated for 10 min at 4°C and centrifuged at 15,000 g for 20 min at 4°C. The supernatant was treated with the RNase A and proteinase K. After DNA was precipitated, the collected DNA was electrophoresed (mentioned above).

Immunohistochemical examination.   To detect the MCP-1 expression and the infiltrating macrophages in heart tissues, we performed an immunohistochemical examination. The sections were deparaffinized and washed in PBS solution. Then, to detect MCP-1, the specimens were incubated with proteinase K (20 µg/ml) for 15 min. After the inhibition of endogenous peroxidase activity with hydrogen peroxide, the sections were incubated with blocking buffer (10% normal goat serum) for 10 min at room temperature. The slides were then incubated with rabbit polyclonal anti-rat MCP-1 antibody (1:20) or mouse monoclonal anti-rat activated macrophage (KiM2R, 1:100, BMA Biomedicals) for 1 h at room temperature. After being washed with PBS, tissue sections were incubated for 1 h with biotinylated goat anti-rabbit IgG (for MCP-1), or biotinylated goat anti-mouse IgG (for KiM2R). Subsequently, sections were washed and incubated for 30 min with peroxidase-conjugated streptavidin. Peroxidase activity was visualized with 3,3'-diaminobenzidine, and the sections were counterstained with hematoxylin. We counted the number of activated macrophages in the ischemic border region and nonischemic region. In addition, to examine whether apoptotic cells express MCP-1 protein, we performed double staining with TUNEL and the MCP-1 antibody. After TUNEL staining, sections were incubated with anti-rat MCP-1 antibody and biotinylated goat anti-rabbit IgG (mentioned above). Then the slides were incubated with alkaliphosphatase-conjugated streptavidin, and alkaliphosphatase activity was visualized with 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (BCIP/NBT). Finally, nuclei were stained with Kernechitrot.

Statistics

All values are expressed as means ± SE. Differences were analyzed by one-way ANOVA combined with Sheffé's multiple comparison test (see Figs. 1, 4, and 5) or the unpaired Student's t-test (see Figs. 2, 3, 6, and 8). A P value <0.05 was considered significant.


Figure 1
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  		Fig. 1. Staurosporine (STS)-induced myocyte apoptosis. A: myocytes were treated with the indicated concentration of STS for 4 h. They were then stained with an anti-myosin heavy chain mono-clonal antibody and DAPI. Arrowheads indicate the apoptotic myocytes. A, inset: the condensed chromatin and fragmented nucleus, which is representative feature of apoptotic nucleus. Bars indicate 25 µm. B: dose-dependent effect of STS on apoptosis. Myocytes were treated with the indicated concentration of STS (0.2–1 µmol/l) for 4 h. The percentage of apoptotic cells was calculated as described in METHODS (n = 6). STS0.2: STS 0.2 µmol/l; STS0.5: STS 0.5 µmol/l; STS1: STS 1 µmol/l. *P < 0.01, {dagger}P < 0.001 vs. control.

 

Figure 4
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  		Fig. 4. Migration of monocytes to the supernatants of cultured apoptotic myocytes. Supernatants of STS-treated myocytes with or without anti-MCP-1 neutralizing antibody were harvested and compared with the supernatant of control myocytes in a microchemotaxis assay for their potential to induce monocyte chemotaxis. Control, myocyte supernatant; STS1, supernatants of STS (1 µmol/l)-treated myocytes; STS1 + MCP-1 antibody, supernatants of STS (1 µmol/l)-treated myocytes with anti-MCP-1 neutralizing antibody; STS1-nonspecific antibody, supernatants of STS (1 µmol/l)-treated myocytes with nonspecific rabbit Ig G; DMEM, fresh medium; DMEM + STS1, fresh medium with STS 1 µmol/l. Values are means of 7 experiments, expressed as percent migration as induced by the control supernatant. **P < 0.0001 vs. control, #P < 0.05 vs. STS1.

 

Figure 5
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  		Fig. 5. IL-1β content of myocytes after STS treatment. Myocytes were incubated in the presence or absence of STS (1 µmol/l) for the indicated time periods. Cell extracts were prepared from the control and STS-treated myocytes, and aliquots were subjected to ELISA for IL-1β as described in METHODS (n = 6). *P < 0.05 vs. control.

 

Figure 2
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  		Fig. 2. Staurosporine (1 µmol/l)-induced myocyte apoptosis. A: electrophoretic analysis of DNA fragmentation. Cardiac myocytes were incubated in the presence or absence of STS (1 µmol/l) for 4 h. The low-molecular-weight genomic DNA was extracted from the myocytes and subjected to agarose gel electrophoresis. M, molecular weight markers. Results are representative of 5 independent experiments. B: caspase-3 activity in cardiac myocytes. Cardiac myocytes were incubated in the presence or absence of STS (1 µmol/l) for 4 h. Caspase-3 activity was measured as described in METHODS (n = 7). {dagger}P < 0.001 vs. control.

 

Figure 3
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  		Fig. 3. Production of monocyte chemoattractant protein-1 (MCP-1) in apoptotic myocytes. A: expression of MCP-1 mRNA after STS treatment. Myocytes were incubated in the presence or absence of STS (1 µmol/l) for 1.5 h. Total RNA was then extracted, and the levels of MCP-1 mRNA were examined by RT-PCR. Data on GAPDH are shown to demonstrate equal loading. Representative results are shown from 6 independent experiments. {dagger}P < 0.001 vs. control. B: level of MCP-1 protein after STS treatment. Myocytes were incubated in the presence or absence of STS (1 µmol/l) for 4 h. Cell extracts were prepared from the control and STS-treated myocytes, and aliquots containing 80 µg of protein were subjected to Western blotting and probed with an antibody to MCP-1 as described in METHODS. Representative immunoblots are shown from 6 independent experiments. Values are expressed as the fold increase above the values found in untreated control cultures (n = 6). *P < 0.05 vs. control. C–F: immunochemical findings of MCP-1 protein after STS treatment. Myocytes were treated with STS (1 µmol/l) for 4 h. C: myocytes were stained with an anti-MCP-1 antibody (green) and DAPI (blue). D: the same myocytes as in C were stained with anti-myosin heavy chain (red) and DAPI (blue). E: control staining omitting anti-MCP-1 antibody. F: the same myocyte as in E were stained with anti-myosin heavy chain (red) and DAPI (blue). An arrowhead indicates the apoptotic myocytes. Representative of 5 separate experiments. Bars, 10 µm.

 

Figure 6
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  		Fig. 6. Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining of cardiac tissues after 30 min of coronary occlusion and 24 h of reperfusion (A–C). TUNEL-positive nuclei appear brown, and hematoxylin staining indicates TUNEL-negative nuclei. An arrowhead indicates apoptotic myocytes. A: nonischemic region. B: ischemic border region. C: control staining of ischemic border region omitting TdT enzyme. Bars, 50 µm. Representative of at least 7 separate experiments. D: ratio of TUNEL-positive nuclei in the ischemic border region (IB) and nonischemic region (NI) (n = 7). *P < 0.0001 vs. NI. E: detection of DNA fragmentation using agarose gel electrophoresis. M, molecular weight markers. Representative of 5 separate experiments.

 

Figure 8
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  		Fig. 8. Double staining using the TUNEL technique and immunohistochemistry with MCP-1 antibody (A and B), and immunohistochemical staining of activated macrophages (C and D) in continuous heart sections. Heart tissues were obtained after ischemia-reperfusion and were processed for immunohistochemical analysis, as described in METHODS. A and B: TUNEL-staining was visualized with 3,3'-diaminobenzidine (brown nuclei) and immunohistochemical staining for MCP-1 was visualized with BCIP/NBT (blue cytosol). C and D: and immunohistochemical staining for activated macrophages was visualized with 3, 3'-diaminobenzidine. An arrowhead indicates MCP-1-expressing apoptotic myocytes, and an arrow indicates activated macrophages. A and C: ischemic border region. B and D: nonischemic region. Bars, 50 µm. Representative of at least 7 separate experiments. E: number of activated macrophages in the ischemic border region and nonischemic region (n = 7). *P < 0.0001 vs. NI.

 

   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
In Vitro Experiments

Induction of myocyte apoptosis.   Nuclear staining with DAPI showed that STS induced the apoptosis of myocytes in a dose-dependent fashion (Fig. 1A). STS (0.5 or 1 µmol/l) significantly increased the percentage of apoptotic myocytes to 9.46 ± 1.02% and 16.4 ± 1.07%, respectively, compared with the serum-deprived control (3.73 ± 0.82%) (Fig. 1B). The histological evidence of apoptosis in STS-treated myocytes was confirmed by the DNA analysis. As shown in Fig. 2A, control myocytes showed rare DNA fragmentation. In contrast, STS (1 µmol/l) caused extensive DNA fragmentation, producing the characteristic DNA ladders. Concomitantly, caspase-3 activity in the myocytes treated with STS (1 µmol/l) also significantly increased by 4.4-fold compared with that in the serum-deprived control (n = 7, P < 0.001) (Fig. 2B). Treatment of myocytes with any dose of STS for 4 h did not increase the release of lactate dehydrogenase into the culture medium (data not shown).

Production of MCP-1 in apoptotic myocytes.   Treatment of myocytes with STS at 1 µmol/l for 1.5 h led to a 2.1-fold increase in MCP-1 gene expression (n = 6, P < 0.01) (Fig. 3A). STS (1 µmol/l) also remarkably increased the level of MCP-1 protein after 4 h of exposure (Fig. 3B). Immunochemical examination revealed that apoptotic myocytes were positive for MCP-1 (Fig. 3, C and D). The myocytes, stained without the anti-MCP-1 antibody as a control, revealed no staining for MCP-1 (Fig. 3, E and F).

Role of MCP-1 in monocyte/macrophage migration.   The culture medium of the myocytes treated with STS (1 µmol/l) for 4 h recruited more monocytes in the trans-well system than did the culture medium of control myocytes (n = 7, P < 0.0001). In addition, the anti-MCP-1 neutralizing antibody, but not nonspecific IgG, significantly attenuated the migration of monocytes. On the other hand, fresh medium containing STS (1 µmol/l) did not recruit monocytes (Fig. 4).

Production of IL-1β in apoptotic myocytes.   To determine the mechanism by which the apoptotic signal pathway enhanced MCP-1 expression, we next examined IL-1β expression, because the IL-1β-converting enzyme is usually activated in apoptotic signaling and IL-1β is a well-known inducer of MCP-1. As shown in Fig. 5, treatment with STS significantly increased the IL-1β content of myocytes in a time-dependent fashion.

We determined whether the production of MCP-1 by apoptotic myocytes occurs in an in vivo model.

In Vivo Experiments

Ischemia-reperfusion-induced myocyte apoptosis.   Figure 6 shows TUNEL staining of the nonischemic region (Fig. 6A) and ischemic border region (Fig. 6B) after ischemia-reperfusion and the results of a quantitative analysis (Fig. 6D). TUNEL-positive nuclei were mainly observed in the ischemic border region. In the nonischemic myocardium, no TUNEL-positive nuclei were observed. TUNEL-positive nuclei were detected in both cardiac myocytes and noncardiac myocytes, such as infiltrating leukocytes and vascular endothelial cells. In the ischemic border region, 17.1 ± 1.1% of myocytes were TUNEL positive. The histological evidence of apoptosis in the ischemic border region was confirmed by the DNA analysis. As shown in Fig. 6E, DNA extracts collected from the ischemic border myocardium showed a distinct laddering pattern.

MCP-1 expression in the heart.   Immunohistochemical analyses demonstrated that MCP-1 protein was rarely detectable in the nonischemic region (Fig. 7A). On the other hand, in the ischemic border region, MCP-1 protein was weakly but remarkably detected in cardiac myocytes as well as vascular endothelial cells (Fig. 7B). In the necrotic region, infiltrating mononuclear cells and vascular endothelial cells expressed MCP-1 (Fig. 7C). These observations suggest that cardiac myocytes express MCP-1 after ischemia-reperfusion.


Figure 7
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  		Fig. 7. Immunohistochemical staining for MCP-1 protein in heart tissues. Heart tissues were obtained after ischemia-reperfusion and were processed for immunohistochemical analysis to detect MCP-1, as described in METHODS. MCP-1 staining was found in vascular endothelial cells (big arrow) and mononuclear cells (small arrow) in the necrotic region and weakly found in cardiac myocytes in the ischemic border region (arrowhead). A: nonischemic region. B: ischemic border region. C: necrotic region. D: control staining of ischemic border region omitting anti-MCP-1 antibody. Bars, 50 µm. Representative of at least 5 separate experiments.

 
MCP-1 expression in apoptotic myocytes.   To determine whether apoptotic myocytes express MCP-1 protein in an in vivo model, double immunohistochemical staining with the MCP-1 monoclonal antibody and TUNEL were conducted (Fig. 8, A and B). Brown nuclei indicate TUNEL-positive nuclei, and a blue cytosol indicates the expression of MCP-1 protein. In the ischemic border region, 69.8 ± 3.9% of TUNEL-positive myocytes expressed MCP-1 protein.

Infiltration of activated macrophages.   We examined the immunohistochemical staining of activated macrophages to determine the region of their infiltration (Fig. 8, C and D). In the nonischemic region, few activated macrophages were observed. On the other hand, the number of activated macrophages was increased in the ischemic border region, where TUNEL-positive myocytes were mainly observed (Fig. 8E).


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present study demonstrates for the first time that myocytes induced to undergo apoptosis by STS express MCP-1 mRNA and protein in vitro. The culture medium of apoptotic myocytes recruited monocytes via a MCP-1-dependent mechanism. In addition, in experiments in vivo, apoptotic myocytes expressed MCP-1 protein after ischemia-reperfusion injury and activated macrophages located in the areas of apoptosis.

The present results suggest that apoptotic myocytes actively recruit monocytes/macrophages with transcriptional regulation. One of the characteristics of apoptosis is fragmented DNA, and so apoptotic nuclei might not be capable of transcription. However, the DNA is damaged late in the apoptotic pathway, and early transcriptional regulation has been reported (8). Therefore, it is possible that apoptotic myocytes produce a chemotactic factor to recruit macrophages. Previously, researches on the release of macrophage chemotactic factor by apoptotic cells have yielded inconsistent findings. Some studies have indicated that apoptotic cells participated in the recruitment of macrophages using endometrial epithelial cells, lymphoma cells, and fibroblasts (17, 29, 37). On the other hand, Witting et. al. (42) found that apoptotic neurons did not release soluble signals that serve to attract microglia, brain macrophages, using a microchemotaxis chamber. The reason for this discrepancy remains unclear, but the density of microglia in brain tissue is higher than that of macrophages in heart tissue, so that apoptotic neurons might be apt to be engulfed by phagocytes. Chemotactic factors may depend on the type of apoptotic and phagocytic cells.

The present study suggests that MCP-1 is one of the chemotactic factors recruiting macrophages in the apoptosis of myocytes. Previous studies have reported that cells dying from apoptosis release thrombospondin 1 (17), S19 ribosomal protein dimer (9, 31), macrophage colony-stimulating factor (29), IL-8 (7, 18), and MCP-1 (1, 3, 27, 44). Among these factors, we examined MCP-1 for three reasons: 1) MCP-1 is reported to be a recruiter of macrophages in some cell types (1, 3, 44), 2) cardiac myocytes express MCP-1 under pathological conditions (28, 36), and 3) MCP-1 attenuates hypoxia-induced cell death in cultured cardiac myocytes (35, 36). In the present study, the expression of the MCP-1 gene and protein was upregulated in apoptotic myocytes. Moreover, using a trans-well chemotaxis assay, we have shown that the supernatant of STS-treated myocytes recruits monocytes, and anti-MCP-1 antibody significantly attenuates this migration. Although we still cannot exclude the possibility of the existence of other soluble factors, these results strongly suggest that MCP-1 is one of the dominant chemoattractant factors derived from apoptotic myocytes.

One possible mechanism by which apoptotic myocytes increase MCP-1 production would be via upregulation of the expression of IL-1β, a well-known inducer of MCP-1 (40). In the present study, the IL-1β content of myocytes increased after STS treatment. Recently, Syed et. al. (33) have reported that the expression of interleukin-converting enzyme (ICE)/caspase-1, a proinflammatory and proapoptotic caspase, is upregulated after myocardial ischemia. Moreover, the activation of caspase-1 is also involved in STS-induced neuronal apoptosis (23). In addition, in the neonatal rodent brain, ischemia rapidly stimulates the expression of MCP-1, and this phenomenon is attenuated with the homozygous deletion of ICE (43). On the basis of these previous findings, apoptotic stimuli would activate ICE/caspase-1, with a subsequent increase in the production of IL-1β, leading to the generation of MCP-1 in the present study.

The present study suggests that apoptotic myocyte-derived MCP-1 has a critical role in the recruitment of macrophages. However, in vivo, we could only demonstrate that apoptotic myocytes expressed MCP-1 and activated macrophages infiltrated in the vicinity of apoptotic myocytes. Therefore, we do not have in vivo evidence that apoptotic myocyte-derived MCP-1 is a critical recruiter of macrophages, and our data cannot exclude other roles for apoptotic myocyte-derived MCP-1 in cardiac tissues. Recently, Zhou et.al. (47) have reported that a functional MCP-1 receptor exists in cardiac myocytes, and prolonged stimulation of this receptor induced myocytes apoptosis. Consequently, apoptotic myocyte-derived MCP-1 might also have had the autocrine effects on the myocytes themselves in the present study. The importance and role of the MCP-1 derived from apoptotic myocytes in cardiac tissue have not been fully elucidated and require further examination. In addition, the present study demonstrated that apoptotic myocytes coexpressed MCP-1 protein in vitro and in vivo and apoptotic stimuli enhanced MCP-1 mRNA expression in cultured myocytes, but did not provide direct evidence of the contribution of apoptotic myocytes to the MCP-1 production. This point should be also elucidated in further research.

In conclusion, the present results suggest that apoptotic myocytes actively generate MCP-1 and mediate the recruitment of monocytes/macrophages. The adequate recruitment of phagocytes associated with apoptosis may contribute to the efficient removal of apoptotic cells.


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This study was supported by a grant from the Ministry of Education, Science, and Culture (No. 13770355) in Japan.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. Kobara, Dept. of Clinical Pharmacology, Kyoto Pharmaceutical Univ., 5 Misasagi Nakauchi-cho, Yamashina-ku, Kyoto, Japan (e-mail: kobara{at}mb.kyoto-phu.ac.jp)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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