ATS
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to Personal Folders
Right arrow Download to citation manager
Right arrow Author home page(s):
Avio M. Perna
Right arrow Permission Requests
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Formigli, L.
Right arrow Articles by Perna, A. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Formigli, L.
Right arrow Articles by Perna, A. M.
Related Collections
Right arrow Myocardial protection

Ann Thorac Surg 2001;71:1596-1602
© 2001 The Society of Thoracic Surgeons

Original article: cardiovascular

Are macrophages involved in early myocardial reperfusion injury?

Lucia Formigli, BSca, Lidia Ibba Manneschi, MDa, Chiara Nediani, BScb, Elena Marcelli, MDd, Geri Fratini, MDc, Sandra Zecchi Orlandini, BSca, Avio M. Perna, MDc

a Department of Anatomy, Histology and Forensic Medicine, University of Florence, Florence, Italy
b Department of Biochemical Sciences, University of Florence, Florence, Italy
c Cardiac Surgery Unit, Careggi Hospital, Florence, Italy
d Biomedical Technology Unit, S. Orsola-Malpighi Hospital, Bologna, Italy

Accepted for publication December 14, 2000.

Address reprint requests to Prof Zecchi-Orlandini, Dipartimento di Anatomia, Istologia, Medicina Legale, V. le Morgagni, 85, 50134 Florence, Italy
e-mail: orlandini{at}unifi.it


   Abstract
Top
 Abstract
Introduction
 Material and methods
 Results
 Comment
 Acknowledgments
 References
 
Background. Neutrophils are the predominant phagocytes in the early stages of myocardial ischemia-reperfusion response and are also implicated in the development of tissue damage. This study examined the role of recruited macrophages in the evolution of this tissue injury.

Methods. Farm pigs were subjected to 30 minutes of myocardial ischemia followed by 30 minutes of reperfusion. Biopsy samples were taken from the control, ischemic, and ischemic-reperfused left ventricle wall and processed for both morphologic and biochemical analyses. In situ production of tumor necrosis factor-{alpha} was evaluated by Western blot and immunofluorescence. A full hemodynamic evaluation was also performed.

Results. Myocardial ischemia and early reperfusion caused marked neutrophil and macrophage tissue accumulation and tumor necrosis factor-{alpha} production by the injured tissue. Immunofluorescence studies allowed us to localize tumor necrosis factor-{alpha} predominantly in tissue-infiltrating macrophages. No depression in the global myocardial contractile function was observed, either during ischemia or after reperfusion.

Conclusions. These data suggest that the newly recruited macrophages within the ischemic and early postischemic myocardium may play a role in promoting neutrophil tissue infiltration and subsequent neutrophil-induced tissue dysfunction by producing tumor necrosis factor-{alpha}.


   Introduction
Top
 Abstract
 Introduction
Material and methods
 Results
 Comment
 Acknowledgments
 References
 
Many studies have focused on the role played by the acute myocardial inflammatory reaction as a mediator of the ischemia-reperfusion syndrome [1]. Strategies aimed at interfering with the generation of inflammatory cytokines [2] or with the process of tissue leukocyte infiltration [3] are able, in fact, to attenuate and even prevent the increased vascular permeability and myocyte destruction associated with reperfusion. Although it has been clearly demonstrated that neutrophils are the predominant leukocytes accumulating in the injured myocardium and the major source of the reactive oxygen metabolites produced at reperfusion, the role of other leukocyte subtypes, namely macrophages, in this injury response remains to be clarified. On the other hand, macrophages become the prevalent cells of the inflammatory infiltrate in the late reperfusion, thus contributing to the healing and remodeling mechanisms after acute myocardial infarction [4]. However, recent evidence suggests that activated macrophages may also be responsible, at least in part, for the pathogenetic changes that follow ischemia-reperfusion. In fact, plasma levels of macrophage activating factor-1 are elevated in patients with acute myocardial infarction [5], and neutralization of this cytokine is beneficial in preventing reperfusion injury [6]. Moreover, it has been demonstrated that tumor necrosis factor-{alpha} (TNF{alpha}) released from activated hepatic macrophages is responsible for the local (liver) and distant (lung) tissue damage by stimulating neutrophil recruitment and activation [7]. Although an involvement of TNF{alpha} in myocardial ischemia-reperfusion injury has been postulated [8–11], the role of myocardial fixed or recruited macrophages as the potential cellular source of this cytokine in the ischemic and ischemic-reperfused myocardium remains to be elucidated. Therefore, it seemed worthwhile to examine, using a model of transient pig myocardium ischemia-reperfusion [12], whether monocytes or macrophages were recruited in the ischemic and early ischemic-reperfused myocardium and whether these cells caused myocardial damage by producing TNF{alpha}.


   Material and methods
Top
 Abstract
 Introduction
 Material and methods
Results
 Comment
 Acknowledgments
 References
 
Surgical protocol
Twelve farm pigs, of either sex, weighing 30 to 50 kg were used for this study. All the animals received humane care according to the guide for the care and use of laboratory animals as stated from European law. Sham-operated control (n = 4), ischemic (I; n = 4) and ischemic-reperfused (I-R; n = 4) pigs were subjected to the temporary occlusion of the distal portion of the left anterior descending coronary artery (LAD), as previously reported [12]. This method allows for the creation of an ischemic zone without hampering the global function of the left ventricle. All the animals were premedicated with intramuscular ketamine (15 mg/kg) and diazepam (5 mg/kg) and anesthetized with sodium pentobarbital (20 mg/kg). Orotracheal intubation was performed, and animals were ventilated using oxygen and fluothane to maintain an arterial oxygen tension greater than 100 mm Hg and a normal pH. The right femoral artery was dissected free and cannulated for arterial pressure measurements. In the I-R animals a full hemodynamic evaluation was also performed. To this purpose, a Millar 5F manometer-tipped catheter (Millar Instruments Inc, Houston, TX) inserted through the carotid artery was used to monitor the ventricular pressure, and an endocardial Best electrode equipped with a microaccelerator (Sorin Instruments, Saluggia, Italy), positioned into the apex of the right ventricle through the right external jugular vein, was used to record the endocardial acceleration, as previously reported [13]. A median sternotomy was then performed in all animals, the pericardium was opened, and the heart was suspended in the pericardial cradle. The LAD was dissected free from the vein and epicardial layer, and encircled with a heavy silk distal to the first major diagonal branch. The LAD was then clamped with a small bulldog clamp, so that an ischemic area corresponding to about one third of the left ventricle was created. The extension of the ischemic area of the left ventricle was 31% ± 5%, as assessed by the triphenyl tetrazolium chloride-Evan’s blue technique [14], which also indicated the absence of an infarcted area within the ischemic region. In particular, triphenyl tetrazolium chloride-Evan’s blue was given with the LAD clamped at the time of hemodynamic evaluation as a 0.3-mL bolus in the proximal LAD using an insulin syringe with an appropriate needle. Myocardial contractility was evaluated by epicardial echocardiography. After 30 minutes of ischemia, a group of animals (I group) was sacrificed and small biopsy samples were taken from the anterior wall of the left ventricle. In the I-R animals, the clamp was released 30 minutes after the induction of ischemia, and reperfusion was performed for a further 30 minutes. The animals were then sacrificed to obtain myocardial fragments from the anterior wall of the left ventricle. Additional myocardial biopsies were taken from sham-operated control pigs. All specimens were then processed for the morphologic and biochemical analysis. Hemodynamic variables, including heart rate, left ventricular pressure maximal rate of rise, cardiac output, systolic pressure, and peak endocardial acceleration, were measured before the coronary occlusion, 20 minutes after the occlusion, and 15 minutes after reperfusion in the I-R animals before sacrifice.

Morphologic analysis
Myeloperoxidase activity
To assess leukocyte infiltration, fragments from the left anterior ventricle wall of each animal were taken and processed to reveal myeloperoxidase activity. The tissue sections were stained with diaminobenzidine (Sigma Chemical Co, St. Louis, MO) and counterstaining was obtained with Mayer’s Haemallum. To determine the number of myeloperoxidase-positive cells an average of 120 random microscopic fields (two for each tissue section) of 138,000 µm2 each were examined in the sham-operated controls, as well as in the I and I-R samples at x250, by two different observers.

Ultrastructural analysis
For the ultrastructural analysis, two myocardial biopsies were taken from the left ventricle wall of each animal. The samples were immediately fixed by immersion in cold glutaraldehyde, postfixed in 1% osmium tetroxide, and routinely processed for transmission electron microscopy. Semithin sections, 2 µm thick, were cut, stained with toluidine blue-sodium tetraborate, and observed under light microscopy. For each specimen, two sets of ultrathin sections were cut at different levels and placed on two different transmission electron microscopy grids of 200 meshes, stained with uranyl acetate and alkalin bismuth subnitrate, and examined under a transmission electron microscope (Jeol Jem 1010, Tokyo, Japan). For each grid, four different randomly chosen openings were observed, and the number of macrophages and neutrophils per grid was calculated at a magnification of x2,000.

Immunofluorescence
To detect the cellular sources of TNF{alpha} production, myocardial samples from sham-operated control, I, and I-R animals were immediately fixed in 4% paraformaldehyde for 4 h and frozen at -80°C until sectioning. Cryostat sections (5 to 6 µm thick) were incubated with 1 : 80 dilution of rabbit polyclonal Ab directed against TNF{alpha} (Calbiochem-Novabiochem, Cambridge, MA), and the immunoreactivity was revealed by fluorescein-conjugated immunoglobulin diluted 1 : 40. Negative controls were obtained by substituting the primary antibody with nonimmune serum. Immunofluorescence was detected using a Nikon Microphot-Fx microscope (Nikon, Tokyo, Japan).

Biochemical analysis
Myocardial biopsy specimens from sham-operated control, I, and I-R animals were rinsed in ice-cold saline solution and frozen at -70°C until use.

Alpha naphthyl acetate esterase assay
To detect the activity of alpha naphthyl acetate esterase, a marker enzyme of monocytes and macrophages, frozen specimens of sham-operated control, I, and I-R animals were separately homogenized in ice-cold 0.25 mol/L sucrose (1 : 5; weight to volume) with an Ultraturrax apparatus for 2 x 5 seconds. The samples were centrifuged at 10,000 g for 10 minutes at 4°C. The supernatant was further sonicated for 90 seconds in ice, centrifuged at 105,000 g for 90 minutes at 4°C, and assayed for protein. An equivalent of 25 µg of supernatant protein for each sample and 5 µL of 200 mmol/L alpha naphthyl acetate (Merk, Darmstadt, Germany), dissolved in 95% ethanol to give a final concentration of 0.5 mmol/L, was added to a final volume of 2 mL of saline solution. Blanks received no substrate. After 10 minutes of incubation at 37°C, the reaction was stopped by adding 116 µL of sodium dodecyl sulfate solution (12.5 g/100 mL distilled water). Subsequently, 5 µL of 200 mmol/L alpha naphthyl acetate was added to the blanks. Finally, 30 µL of fast red solution (10 mg/mL distilled water; Fast Red B, Sigma Chemical) was added to the sample, followed by an incubation period of 15 minutes at room temperature. Optical density absorption at 490 nm was used to estimate the metabolism of alpha naphthyl acetate to alpha naphthol. Alpha naphthyl acetate esterase activity was estimated as absorption at 490 nm per 25 µg of protein.

Western blot analysis of tumor necrosis factor-{alpha}
Frozen cardiac specimens of sham-operated control, I, and I-R animals were separately homogenized with Ultraturrax apparatus 2 x 15 seconds in a lysis buffer containing 62.5 mmol/L ethylenediaminetetraacetic acid, 50 mmol/L Tris-HCl, pH 8.0, 0.4% deoxycholic acid, 1% Nonidet p-40, 10 µg/mL leupeptin, 10 µg/mL aprotinin, 1 mmol/L phenylmethansulfonylfluoride. The homogenates were centrifuged at 21,000 g for 10 minutes at 4°C, and the supernatants were assayed for proteins. Proteins were eluted from the supernatant directly into sodium dodecyl sulfate sample buffer for 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane in a buffer containing 25 mmol/L Tris-HCl, pH 8.3, 192 mmol/L glycine, 20% methanol. Membranes were blocked in buffer containing 50 mmol/L Tris-HCl, pH 7.4, 150 mmol/L NaCl, 0.1% Tween-20, and 5% bovine serum albumin for 2 hours at room temperature, and incubated with polyclonal antibody against human recombinant TNF{alpha} (Calbiochem, Novabiochem, Cambridge, MA) diluted 1 : 800 overnight at 4°C. Immunodetection of the anti-TNF{alpha} antibody was performed with 1 : 20,000 diluted peroxidase-conjugated anti-rabbit immunoglobulin (Sigma Chemical) for 1 hour at room temperature, and the signal was visualized using enhanced chermoluminescence (ECL) detection reagents (Amersham Pharmacia, Milan, Italy) and Biomax Light-1 film (Eastman Kodak Co, Rochester, NY). Recombinant human TNF{alpha} (Sigma Chemical) was used as a positive control. Signals were quantified using the program for image analysis and densitometry quantiscan (Biosoft, Cambridge, UK).

Statistical analysis
The results of the analysis were indicated as mean ± standard error of the mean. Statistical analysis of the data was performed with two-tailed Student’s t test for unpaired values. Differences were considered statistically significant at p less than 0.05.


   Results
Top
 Abstract
 Introduction
 Material and methods
 Results
Comment
 Acknowledgments
 References
 
Echocardiography
By epicardial echocardiography evaluation, no substantial alteration of the myocardial contractility was observed.

Hemodynamic variables
Table 1 lists the values of systolic pressure, left ventricular pressure maximal rate of rise, heart rate, cardiac output, and peak endocardial acceleration as evaluated before the occlusion, during ischemia, and on reperfusion in the I-R group of animals. Systolic pressure did not significantly change with respect to the baseline values both during ischemia and on reperfusion. Similarly, heart rate remained substantially the same in the course of ischemia, whereas it increased an average of 18 beats/min after reperfusion. On the contrary, left ventricular pressure maximal rate of rise increased to more than the baseline values both during ischemia and reperfusion, and these results were well matched with those of peak endocardial acceleration. Finally, cardiac output was raised during ischemia and further increased after reperfusion.


View this table:
[in this window]
[in a new window]
  		Table 1. Hemodynamic Variables

 
The above-reported hemodynamic variables recorded in sham-operated control animals did not show any significant variation when compared with the baseline values.

Myeloperoxidase activity
The myeloperoxidase activity performed on myocardial paraffin-embedded tissue sections was significantly increased during ischemia and early reperfusion (9.3 ± 1.16 and 28.7 ± 2.6 positive cells/microscopic field, respectively) compared with that of controls (3.1 ± 0.56, p < 0.001; Fig 1). Myeloperoxidase-positive cells were observed both in the lumen of small blood vessels and in the interstitial spaces among cardiomyocytes.



View larger version (44K):
[in this window]
[in a new window]
  		Fig 1. Myeloperoxidase (MPO) activity in sham-operated control, ischemic (I), and ischemic-reperfused (I-R) pig myocardium. °p < 0.001 versus control myocardium; *p < 0.001 versus ischemic (I) myocardium.

 
Alpha naphthyl acetate esterase
The presence of tissue monocytes within the inflammatory infiltrate of the ischemic and early reperfused myocardium was revealed by determination of the activity of a specific monocyte-derived enzyme, alpha naphthyl acetate esterase (0.4 ± 0.06 and 1.03 ± 015, respectively, versus 0.17 ± 0.04 of the sham-operated control samples; p < 0.001; Fig 2).



View larger version (46K):
[in this window]
[in a new window]
  		Fig 2. Alpha naphthyl acetate esterase (ANAE) activity in sham-operated control, ischemic (I), and ischemic-reperfused (I-R) hearts. Alpha naphthyl acetate activity was measured on 25-µg protein samples of cytosolic preparations at a final alpha naphthyl acetate concentration of 0.5 mmol/L. Values are mean ± standard error of the mean. p < 0.05 by Student’s t test. °p < 0.001 versus control myocardium; *p < 0.001 versus ischemic (I) myocardium. (ANAE activity is expressed as absorbance at 490 nm per 25 µg protein).

 
Morphologic analysis
Light and transmission electron microscopy allowed the identification of neutrophils and monocytes as the major cellular components of the leukocyte accumulation of the I and I-R myocardium (Fig 3). Indeed, circulating neutrophils and monocytes adhered to the endothelium of small blood vessels (Figs 4a, 4b) and emigrated through the interendothelial junctions and in the interstitial matrix (Figs 4c, 4d). To verify the occurrence of a peripheral recruitment of these cells, we performed an average monocyte and neutrophil count on ultrathin sections from control, I, and I-R samples. The number of both neutrophils and monocytes rose significantly both during ischemia and reperfusion (Table 2). In particular, ischemia and reperfusion caused, respectively, a twofold and eightfold increase in the number of tissue infiltrating neutrophils with respect to that of sham-operated controls. The increase in the number of recruited monocytes was even more pronounced, rising threefold during ischemia and 12-fold on reperfusion. Concomitant with myocardial leukocyte infiltration, ultrastructural signs of tissue damage could also be observed. During ischemia, some cardiomyocytes contained swollen mitochondria with disrupted cristae and showed moderate intermyofibrillar edema (Fig 5a). At reperfusion, these modifications were more pronounced and associated with focal areas of hypercontraction (Fig 5b) and overstretching of myofilaments (Fig 5c). Sometimes, the endothelium of small blood vessels was composed of swollen endothelial cells with loss of plasmamembrane integrity (Fig 5d). Normal sham-operated myocardium showed a quite normal morphology with minimal presence or even absence of tissue infiltrating neutrophils.



View larger version (111K):
[in this window]
[in a new window]
  		Fig 3. Light microscopic examination of ischemic-reperfused myocardium. Monocytes (arrows) are present together with neutrophils within the inflammatory infiltrate. (Toluidine blue sodium-tetraborate stained sections, x800.)

 


View larger version (189K):
[in this window]
[in a new window]
  		Fig 4. Transmission electron microscopy of ischemic-reperfused myocardium showing the various steps of monocyte extravasion. Monocytes are visible (a) inside a small blood vessel (arrows) together with neutrophils (arrowheads); (b) and (c) tightly adhering to the vascular luminal surface (arrows); and (d) migrating in the interstitial spaces (arrowhead). (a: x1,500; b: x11,250; c: x4,500; d: x2,250.)

 

View this table:
[in this window]
[in a new window]
  		Table 2. Monocyte and Neutrophil Count

 


View larger version (187K):
[in this window]
[in a new window]
  		Fig 5. Transmission electron microscopy of ischemic (a) and ischemic-reperfused (b–d) myocardium. (a) Signs of mild injuries, ie, moderate mitochondria swelling (arrows) and intermyofibrillar edema are visible inside a cardiomyocyte; (b) severe hypercontractions (arrows) of myofilaments are seen in one fiber; (c) higher magnification of a cardiomyocyte showing dark myofibrillar material interspersed with overstretched myofilaments; and (d) a capillary is lined by a seriously affected endothelial cell (arrowheads indicate the endothelial lining and arrow shows the nucleus of the endothelial cell). (a: x3,700; b: x3,700; c: x5,500; d: x4,500.)

 
Tumor necrosis factor-{alpha} release
Western blot analysis of TNF{alpha} protein normalized per total proteins or ß-actin (not shown) revealed no signal at the 17-kd position in the sham-operated control myocardium. However, its expression rose versus the controls after ischemia and significantly increased after 30 minutes of reperfusion (3.5-fold versus ischemia), as shown in the densitometric analyses (Fig 6). Immunofluorescence studies performed to identify the sites of TNF{alpha} production showed that an intense TNF{alpha} immunoreactivity was predominantly localized in the infiltrating monocytes (Fig. 7). No reactivity was detected in any other cell type found in the myocardium, except for a weak TNF{alpha} staining in the endothelium of small blood vessels. No specific staining was observed in the control samples (data not shown).



View larger version (35K):
[in this window]
[in a new window]
  		Fig 6. (a) Western blot analysis of tumor necrosis factor-{alpha} (TNF{alpha}) in myocardial tissues from sham-operated control (C), ischemic (I), and ischemic-reperfused (I-R) hearts. Myocardial samples of 50 µg of protein were applied per slot. Tumor necrosis factor-{alpha} protein was expressed as a 17-kd band. Recombinant human TNF{alpha} was used as positive control (PC). (b) Quantitative data from Western blot analysis. Each bar represents the mean value + standard error of the mean of four different blots. Signals were quantified by densitometric analyses and were expressed as densitometric units per microgram of protein. *p < 0.05 I-R versus I.

 


View larger version (22K):
[in this window]
[in a new window]
  		Fig 7. Immunofluorescence staining of ischemic-reperfused myocardium with polyclonal antibody against tumor necrosis factor-{alpha}. Numerous macrophages, infiltrating the connective tissue among cardiomyocytes, are markedly stained. (x500.)

 

   Comment
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
Acknowledgments
 References
 
A substantial body of evidence indicates that myocardial ischemia and, in particular, ischemia plus reperfusion induce a marked neutrophil tissue infiltration [15]. These cells play an important role in the evolution of the tissue alterations by producing superoxide anions and releasing lysosomal enzymes [1–3], as soon as oxygen is restituted to the tissue. The results of the present study implicate another cell type in the myocardial leukocyte recruitment within the ischemic and early reperfused myocardium that is the mononuclear phagocyte. Indeed, although neutrophils were the prominent cells of the inflammatory infiltrate, a significant increase in the number of macrophages was observed after both ischemia and reperfusion, suggesting a peripheral recruitment of these cells within the injured myocardium. Leukocyte influx during ischemia is in agreement with previous observations showing neutrophil infiltration and activation within porcine myocardium after acute ischemia [16, 17].

The existence of a coronary collateral blood flow in pig hearts, which is required for the neutrophils and monocytes tissue accumulation observed in our samples, has been verified by experimental evidence [18–20]. In addition, we have recently shown that the clamping of the LAD, in pigs undergoing coronary artery bypass grafting, has to be performed both above and below the sites of arterotomy to avoid the presence of blood flow downstream impairing the construction of the anastomoses (Perna, personal communication).

In particular, the ultrastructural analysis documented all the steps of monocyte extravasation occurring during ischemia and on reperfusion, showing monocytes closely adhering to the vascular endothelium, migrating through the endothelial cell junctions, and accumulating in the myocardial interstitial spaces. The extensive myocardial leukocyte sequestration correlated well with the appearance of focal injuries to the vessel wall and neighboring parenchyma on reperfusion. It is probable that the mild injuries observed in the ischemic cardiomyocytes may be caused by a decreased adenosine triphosphate (ATP) availability after coronary occlusion. However, the morphologic changes observed both during ischemia and on reperfusion were unable to affect the local myocardial contractile function as shown by epicardial echocardiography findings and by the values of peak endocardial acceleration. In addition, the hemodynamic variables recorded from the I and I-R areas remained unchanged or increased with respect to those found before the coronary occlusion, suggesting the absence of myocardial irreversible damage at least for the time intervals considered in our experimental protocol.

The presence of a conspicuous macrophage infiltration in the I and early reperfused myocardium, in the absence of an infarcted area, raised the question of whether these cells could play a role in the pathogenesis of myocardial reperfusion injury. This hypothesis is further substantiated by the well-accepted idea that macrophages represent the prominent cellular source of TNF{alpha} [21], a pleomorphic cytokine with multiple effects on neutrophil functions. Indeed, numerous in vivo and in vitro studies have demonstrated that TNF{alpha} increases the expression on the vascular endothelium as well as on cardiomyocytes of specific endothelial adhesion proteins for neutrophils, including E-selectin and intercellular adhesion molecule 1 [22], and causes the activation of neutrophils [23]. Moreover, an involvement of macrophage-derived TNF{alpha} in the process of neutrophil tissue accumulation has been revealed in several experimental models of ischemia-reperfusion [7], including hepatic ischemia-reperfusion and hepatic and intestinal ischemia-reperfusion–induced pulmonary dysfunctions [7, 24]. Interestingly, our experiments indicate that TNF{alpha} was produced during myocardial ischemia and particularly on reperfusion and that the site of its synthesis was mainly represented by the tissue-infiltrating macrophages. The importance of ischemia and ischemia-reperfusion in eliciting TNF{alpha} production was emphasized by the fact that the normal myocardium was negative for this cytokine. Although the contribution of other cell types, namely cardiomyocytes [25] and cardiac mast cells [15] in the production of this cytokine cannot be excluded, we were unable to detect TNF{alpha} immunoreactivity in these cells in the reperfused myocardium. On the other hand, the intense immunostaining of TNF{alpha} observed in the tissue-recruited macrophages certainly suggests that these cells may represent a significant source of TNF{alpha} at least in our experimental conditions. Consistent with this hypothesis is recent evidence indicating that porcine myocardial macrophages seem to be the only cell type expressing TNF{alpha} after a period of ischemia [26]. From our findings, it is reasonable to speculate that macrophages infiltrating the ischemic and early reperfused myocardium may play a role in the evolution of the tissue dysfunctions, exerting, through the production of TNF{alpha}, a modulating influence on myocardial neutrophil accumulation. However, a more direct role for macrophage-derived TNF{alpha} in mediating the tissue injuries cannot be ruled out, as it has been recently demonstrated that this cytokine promotes a direct depression of contractility and induces apoptosis in postischemic cardiomyocytes [26].

In conclusion, the results of the present study indicate that a consistent macrophage infiltration occurs during myocardial ischemia and early reperfusion and suggest for these cells an active role in neutrophil tissue recruitment. Studies aimed at blocking the process of macrophage accumulation are in progress in our laboratory to better clarify the role of these cells in promoting reperfusion myocardial damage.


   Acknowledgments
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
 Acknowledgments
References
 
This work was supported by the Ministero della Ricerca Scientifica e Tecnologica (MURST 60%) to Lucia Formigli, Lidia Ibba Manneschi and Sandra Zecchi Orlandini. The authors are grateful to Dr Alessia Tania and Ferdinando Paternostro for their valuable technical assistance in the histologic and statistical analyses.


   References
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
 Acknowledgments
 References
 

  1. Zahler S., Massoudy P., Hartl H.F., et al. Acute cardiac inflammatory responses to postischemic reperfusion during cardiopulmonary bypass. Cardiovasc Res 1999;41:722-730.[Abstract/Free Full Text]
  2. Crawford M.H., Grover F.L., Kolb W.P., et al. Complement and neutrophil activation in the pathogenesis of ischemic myocardial injury. Circulation 1988;78:1449-1458.[Abstract/Free Full Text]
  3. Altavilla D., Squadrito F., Ioculano M., et al. E-selectin in the pathogenesis of experimental myocardial ischemia-reperfusion injury. Eur J Pharmacol 1994;270:45-51.[Medline]
  4. Trial J., Baughn R.E., Wygant J.N., et al. Fibronectin fragments modulate monocyte VLA-5 expression and monocyte migration. J Clin Invest 1999;104:419-430.[Medline]
  5. Matsumori A., Furukawa Y., Hashimoto T., et al. Plasma levels of the monocyte chemotactic and activating factor/monocyte chemoattractant protein-1 are elevated in patients with acute myocardial infarction. J Mol Cell Cardiol 1997;29:419-423.[Medline]
  6. Ono K., Matsumori A., Furukawa Y., et al. Prevention of myocardial reperfusion injury in rats by an antibody against monocyte chemotactic and activating factor/monocyte chemoattractant protein-1. Lab Invest 1999;79:195-203.[Medline]
  7. Colletti L.M., Kunkel S.L., Walz A., et al. Chemokine expression during hepatic ischemia/reperfusion-induced lung injury in the rat. J Clin Invest 1995;95:134-141.
  8. Eddy L.J., Goeddel D.V., Wong G.H.W. Tumor necrosis factor-{alpha} pretreatment is protective in a rat model of myocardial ischemia-reperfusion injury. Biochem Biophys Res Commun 1992;184:1056-1059.[Medline]
  9. Squadrito F., Altavilla D., Zingarelli B., et al. Tumor necrosis factor involvement in myocardial ischaemia-reperfusion injury. Eur J Pharmacol 1993;237:223-230.[Medline]
  10. Gurevitch J., Frolkis I., Yuhas Y., et al. Anti-tumor necrosis factor-alpha improves myocardial recovery after ischemia and reperfusion. J Am Coll Cardiol 1997;30:1554-1561.[Abstract]
  11. Aukrust P., Ueland T., Lien E., et al. Cytokine network in congestive heart failure secondary to ischemic or idiopathic dilated cardiomyopathy. Am J Cardiol 1999;83:376-382.[Medline]
  12. Nediani C., Perna A.M., Liguori P., et al. Beneficial effect of the 21-aminosteroid U 74389G on the ischemia-reperfusion damage in pig hearts. J Mol Cell Cardiol 1997;29:2825-2835.[Medline]
  13. Padeletti L, Perna AM, Michelucci A, et al. Contractility and peak endocardial acceleration (PEA) during temporary coronary occlusion in pigs [Abstract]. New Frontiers of Arrhythmia, Marilleve, Feb 24–27, 1998:136.
  14. Klein H.H., Pushmann S., Shaper J., Shaper W. The mechanism of the tetrazolium reaction in identifying experimental myocardial infarction. Arch Pathol Anat 1981;393:287-297.
  15. Frangogiannis N.G., Lindsey M.L., Michael L.H., et al. Resident cardiac mast cells degranulate and release preformed TNF-{alpha}, initiating the cytokine cascade in experimental canine myocardial ischemia/reperfusion. Circulation 1998;98:699-710.[Abstract/Free Full Text]
  16. Luo W.S., Guo Z.G., Tang X.L. Neutrophil infiltration in ischemic porcine myocardium and protective effects of verapamil. Chung Kuo Yao Li Hsueh Pao 1990;11:435-438.
  17. Curtis W.E., Gillinov A.M., Wilson I.C., et al. Inhibition of neutrophil adhesion reduces myocardial infarct size. Ann Thorac Surg 1993;56:1069-1073.[Abstract]
  18. Maxwell M.P., Hearse D.J., Yellon D.M. Species variation in the coronary collateral circulation during regional myocardial ischaemia: a critical determinant of the rate of evolution and extent of myocardial infarction. Cardiovasc Res 1987;21:737-746.[Medline]
  19. Cheng D.C., Jiang M.T., Asokumar B., Boylen P., Sandler A. Comparison of nifedipine and metoprolol on collateral coronary blood flow in a swine model of chronic coronary obstruction and acute ischaemia during isoflurane anaesthesia. Can J Anaesth 1996;43:160-168.[Medline]
  20. Christian T.F., O’Connor M.K., Schwartz R.S., Gibbons R.J., Ritman E.L. Technetium-99m MIBI to assess coronary collateral flow during acute myocardial infarction into two closed-chest animal models. J Nucl Med 1997;38:1840-1846.[Abstract/Free Full Text]
  21. Hofsli E., Bakke O., Nonstad U., Espevik T. A flow cytometric and immunofluorescence microscopic study of tumor necrosis factor production and localization in human monocytes. Cell Immunol 1989;122:405-415.[Medline]
  22. Kamici R., Karliner J.S., Koudssi F., Long C.S. Expression and regulation of adhesion molecules in cardiac cells by cytokines: response to acute hypoxia. Circ Res 1998;82:576-586.[Abstract/Free Full Text]
  23. Larrick J.W., Graham D., Toy K., Lin L.S., Senyk G., Fendly B.M. Recombinant tumor necrosis factor causes activation of human granulocytes. Blood 1987;69:640-644.[Abstract/Free Full Text]
  24. Caty M.G., Guice K.S., Oldham K.T., Remick D.G., Kunkel S.L. Evidence for tumor necrosis factor-induced pulmonary microvascular injury after intestinal ischemia-reperfusion injury. Ann Surg 1990;212:694-700.[Medline]
  25. Meldrum D.R., Cleveland J.C., Jr, Cain B.S., Meng X., Harken A.H. Increased myocardial tumor necrosis factor {alpha} in a crystalloid-perfused model of cardiac ischemia-reperfusion injury. Ann Thorac Surg 1998;65:439-443.[Abstract/Free Full Text]
  26. Arras M., Stasser R., Mohori M., et al. Tumor necrosis factor-alpha is expressed by monocytes/macrophages following cardiac microembolization and is antagonized by cyclosporine. Basic Res Cardiol 1998;93:97-107.[Medline]



This article has been cited by other articles:


Home page
 EndocrinologyHome page
Y. Wen, J. Gu, S.-L. Li, M. A. Reddy, R. Natarajan, and J. L. Nadler
Elevated Glucose and Diabetes Promote Interleukin-12 Cytokine Gene Expression in Mouse Macrophages
Endocrinology, May 1, 2006; 147(5): 2518 - 2525.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
C. A. Reichel, A. Khandoga, H.-J. Anders, D. Schlondorff, B. Luckow, and F. Krombach
Chemokine receptors Ccr1, Ccr2, and Ccr5 mediate neutrophil migration to postischemic tissue
J. Leukoc. Biol., January 1, 2006; 79(1): 114 - 122.
[Abstract] [Full Text] [PDF]

Home page
 CirculationHome page
I. Kondo, K. Ohmori, A. Oshita, H. Takeuchi, J. Yoshida, K. Shinomiya, S. Fuke, T. Suzuki, K. Mizushige, and M. Kohno
Leukocyte-Targeted Myocardial Contrast Echocardiography Can Assess the Degree of Acute Allograft Rejection in a Rat Cardiac Transplantation Model
Circulation, March 2, 2004; 109(8): 1056 - 1061.
[Abstract] [Full Text] [PDF]

Home page
 CirculationHome page
N. Kajihara, S. Morita, T. Nishida, H. Tatewaki, M. Eto, K. Egashira, and H. Yasui
Transfection With a Dominant-Negative Inhibitor of Monocyte Chemoattractant Protein-1 Gene Improves Cardiac Function After 6 Hours of Cold Preservation
Circulation, September 9, 2003; 108(90101): II-213 - 218.
[Abstract] [Full Text] [PDF]

Home page
 J CARDIOVASC PHARMACOL THERHome page
M. Singh and H. K. Saini
Resident Cardiac Mast Cells and Ischemia-Reperfusion Injury
Journal of Cardiovascular Pharmacology and Therapeutics, June 1, 2003; 8(2): 135 - 148.
[Abstract] [PDF]

Home page
 Ann. Thorac. Surg.Home page
D. A. Bull and J. Maurer
Aprotinin and preservation of myocardial function after ischemia-reperfusion injury
Ann. Thorac. Surg., February 1, 2003; 75(2): S735 - 739.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to Personal Folders
Right arrow Download to citation manager
Right arrow Author home page(s):
Avio M. Perna
Right arrow Permission Requests
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Formigli, L.
Right arrow Articles by Perna, A. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Formigli, L.
Right arrow Articles by Perna, A. M.
Related Collections
Right arrow Myocardial protection

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
ANN THORAC SURG ASIAN CARDIOVASC THORAC ANN EUR J CARDIOTHORAC SURG
J THORAC CARDIOVASC SURG ICVTS ALL CTSNet JOURNALS