Journal of Leukocyte Biology Myeloid cells, immune suppression, tumor immunology
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Originally published online as doi:10.1189/jlb.0703358 on January 23, 2004

Published online before print January 23, 2004
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(Journal of Leukocyte Biology. 2004;75:828-835.)
© 2004 by Society for Leukocyte Biology

Delay of neutrophil apoptosis in acute coronary syndromes

C. D. Garlichs*,1,2, S. Eskafi*,1, I. Cicha*, A. Schmeisser{dagger}, B. Walzog{ddagger}, D. Raaz*, C. Stumpf*, A. Yilmaz*, J. Bremer*, J. Ludwig* and W. G. Daniel*

* Medical Clinic II, Friedrich-Alexander-University of Erlangen-Nürnberg, Germany;
{dagger} Heart Center, Dresden, Germany; and
{ddagger} Department of Physiology, Ludwig-Maximilians-University, Munich, Germany

2Correspondence: Medical Clinic II, Friedrich-Alexander-University of Erlangen-Nürnberg, c/o David-Morgenstern-Weg 14, 91056 Erlangen, Germany. E-mail: Christoph.Garlichs{at}rzmail.uni-erlangen.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Apoptosis of polymorphonuclear neutrophils (PMN) is currently discussed as a key event in the control of inflammation. This study determined PMN apoptosis and its underlying mechanisms in controls (C), patients with stable (SAP) or unstable angina (UAP), and with acute myocardial infarction (AMI). Blood was drawn from 15 subjects of each C, SAP, UAP, and AMI. Apoptosis was measured by flow cytometry in isolated PMN (propidium iodide staining) and PMN from whole blood (CD16, Fc{gamma}RIII). Serum cytokines were determined by enzyme-linked immunosorbent assay. Apoptosis of isolated PMN was delayed significantly in acute coronary syndromes (ACS) as compared with SAP or C (C, 51.2±12.6%; SAP, 44.9±13.6%; UAP, 28.4±10.1%; AMI, 20.3±8.5%; AMI or UAP vs. SAP or C, P<0.001). These results were confirmed by measurement of PMN apoptosis in cultured whole blood from patients and controls. Moreover, serum of patients with ACS markedly reduced apoptosis of PMN from healthy donors. Analysis of patients’ sera revealed significantly elevated concentrations of tumor necrosis factor {alpha}, interferon-{gamma} (IFN-{gamma}), granulocyte macrophage-colony stimulating factor (GM-CSF), and interleukin (IL)-1ß in ACS (vs. C and SAP). IFN-{gamma}, GM-CSF, and IL-1ß significantly delayed PMN apoptosis in vitro. Furthermore, coincubation of PMN with adenosine 5'-diphosphate-activated platelets significantly inhibited PMN apoptosis as compared with coculture with unstimulated platelets. This study demonstrates a pronounced delay of PMN apoptosis in UAP and AMI, which may result from increased serum levels of IFN-{gamma}, GM-CSF, and IL-1ß and from enhanced platelet activation. Therapeutical modulation of these determinants of PMN lifespan may provide a new concept for the control of inflammation in ACS.

Key Words: inflammation • unstable angina • acute myocardial infarction • leukocytes


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Inflammation plays a key role in the development and progression of atherosclerosis and its complications [1 ]. Patients with coronary artery disease (CAD) show local inflammation within coronary arteries, which is accompanied by a systemic inflammatory response reflected by the increase of acute-phase proteins and white blood cells (WBC) [2 3 4 ]. WBC seem to play an active, pathogenic role in atherosclerosis, as they exert diverse proatherogenic and prothrombotic effects [5 , 6 ]. Thereby, elevation or activation of leukocytes indicates increased risk of subsequent cardiovascular events and mortality [4 , 7 , 8 ].

Among leukocytes, polymorphonuclear neutrophils (PMN) substantially contribute to the risk for CAD [9 , 10 ]. Currently, their specific role in the pathogenesis of atherosclerosis and thrombosis is an object of intense research [11 ]. It is known that activated PMN produce and release reactive oxygen species (ROS), inflammatory leukotrienes, and proteolytic lysosomal enzymes, which can directly induce vascular damage. Furthermore, in advanced stages of CAD, characterized by frequent occurrence of intracoronary thrombus formation, PMN actively regulate local thrombotic and hemostatic processes [6 ]. Animal models of myocardial infarction demonstrated that PMN depletion, pharmacological suppression of PMN activation, as well as inhibition of PMN-endothelial cell adhesion reduced the extent of acute tissue injury and mortality following ischemia and reperfusion [12 ].

Until now, only a few studies have evaluated PMN functions in advanced CAD, i.e., acute coronary syndromes [ACS; i.e., unstable angina pectoris (UAP) and acute myocardial infarction (AMI)]. In these patients, Takeshita et al. [2 ] found priming of circulating PMN for the generation of oxygen species as a marker of PMN activation, as compared with PMN from patients with stable angina (SAP) [2 ]. In contrast, Mazzone et al. [13 ] reported a transcardiac gradient of activated PMN measured by CD11b/CD18 expression in UAP but no systemic activation of PMN [14 ]. Given the proatherogenic and prothrombotic role of PMN, particularly in the exacerbated inflammatory milieu of ACS, it would therefore be of interest to better characterize functions of circulating PMN in patients with ACS.

To address this topic, we evaluated spontaneous PMN apoptosis and its regulation in patients with ACS, as the induction or prevention of PMN apoptosis is currently discussed as a key event in the control of inflammation [15 16 17 ]. PMN are short-lived, terminally differentiated cells. Their apoptosis limits their histotoxic potential, as apoptotic PMN are phagocytosed by macrophages without releasing their mediators. It also restricts PMN effector function, as it is associated with reduced migration, phagocytosis, degranulation, and reactive oxygen species (ROS) production. The control of PMN lifespan thus contributes to the maintenance of a balance between the potency of the inflammatory response and the risk of tissue damage [15 ].

Although PMN apoptosis seems to be critical for homeostasis as well as for the control of inflammatory processes, until now, no study has evaluated PMN apoptosis in the conditions of ACS. We demonstrate here that patients with ACS show a significant delay of PMN apoptosis as compared with patients with stable angina or healthy controls. Inflammatory cytokines as well as activated platelets seem to be responsible for this phenomenon.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patient selection
Patients were consecutively registered between January 1999 and November 2000. All patients had angiographically proven coronary artery disease (stenosis of >75% in at least one coronary vessel). From among the patients fulfilling the criteria for UAP, 15 consecutive patients were selected to participate in our study (Table 1 ). UAP was defined as rest pain occurring within 48 h preceding the recruitment for the study, without a recent myocardial infarction (Braunwald class IIIB). These patients had no evidence of "major" myocardial necrosis, which is reflected by elevated levels of creatine kinase (CK) or CK-MB isoenzyme, but some of them had "minor" myocardial injury detected by repeated measurements of troponin I (Braunwald class IIIB-Tpositive vs. IIIB-Tnegative) [18 ]. Transient ST-T segment depression and/or T-wave inversion were frequently present in the group of UAP. Furthermore, the study included 15 patients with AMI. Inclusion criteria here were typical anginal pain lasting >30 min, ST-segment elevation of >=1 mm in >=two contiguous leads, and elevation of CK to >=three times the normal upper limit with a concomitant rise in MB isoenzyme. Patients with AMI had to meet at least two of the three above criteria. Blood from patients with UAP or AMI was drawn immediately after their admission to the hospital.


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  		Table 1. Baseline Characteristics of Control Subjects and Patients with SAP, UAP, and AMI

 
During the same period of time, 15 sex- and age-matched patients with SAP were consecutively selected among patients undergoing elective diagnostic coronary angiography or percutaneous coronary angioplasty. All of these latter patients had long-term (>6 months), stable effort angina and a positive exercise test. The control group consisted of 15 sex- and age-matched subjects with atypical chest pain, who underwent coronary angiography for exclusion of coronary artery disease. Exclusion criteria for all groups were interfering noncardiac disease, such as anemia, infection, malignoma, collagen disease, thyrotoxicosis; cardiac disease other than coronary artery disease, except for minor mitral regurgitation; overt right or left ventricular failure; coronary artery bypass surgery, balloon angioplasty, or thrombolytic therapy within 3 months preceding the study. Blood from patients with SAP and controls was collected briefly after admission to the hospital. Premedication in patients with SAP consisted of aspirin; in patients with ACS, aspirin and unfractionized heparin. The blood for some marked in vitro experiments was obtained from healthy volunteers, who had no clinical signs of atherosclerosis and who had not received any medication for at least 2 weeks.

The local ethics committee for human subjects approved the study. Informed consent was obtained from all patients.

Reagents and antibodies
RPMI 1640, fetal calf serum, penicillin/streptomycin, L-glutamine, Dulbecco’s phosphate-buffered saline (PBS), and ethylenediaminetetraacetate (EDTA) were obtained from Biochrom (Berlin, Germany). Percoll was from Pharmacia (Freiburg, Germany). Recombinant human interleukin-1ß (rhIL-1ß), adenosine 5'-diphosphate (ADP) sodium salt, propidium iodide (PI), glucose, bovine serum albumin, Triton X-100, paraformaldehyde, and aspirin were purchased from Sigma (Deisenhofen, Germany). Tumor necrosis factor {alpha} (TNF-{alpha}), rh interferon-{gamma} (IFN-{gamma}), and rh granulocyte macrophage-colony stimulating factor (GM-CSF) were obtained from Biosource (Solingen, Germany). Fluorescein isothiocyanate (FITC)-conjugated mouse monoclonal antibody [mAb; CB16, immunoglobulin G1 (IgG1)] anti-human Fc{gamma}RIII (CD16), phycoerythrin (PE)-conjugated mouse mAb (PM6/13, IgG1) anti-human CD61 [polymorphism of glycoprotein IIIa (GP IIIa)], and PE-conjugated isotype control of mouse IgG1 were obtained from Dianova (Hamburg, Germany). PE-conjugated mouse mAb (6.7, IgG1) anti-human CD18, FITC-conjugated mouse mAb (AK-4, IgG1) anti-human P-selectin (CD62P), and FITC- and PE-conjugated isotype control of mouse IgG1 were obtained from PharMingen (Heidelberg, Germany).

Blood-sampling protocol
Peripheral venous blood was drawn into sodium citrate Monovette (106 mM, 1:10 v/v), lithium heparin Monovette (15 IE/ml blood), and serum Monovette (Sarstedt, Germany) and immediately transferred to the laboratory. Citrate plasma and serum (after clotting of blood) were obtained by centrifugation at 1500 g for 10 min. Serum and plasma were collected and aliquoted under sterile conditions and were stored at –80°C until analysis. The blood collection generally occurred before administration of drugs, except heparin and aspirin.

PMN isolation and culture
PMN were isolated from heparinized blood by Percoll gradient centrifugation as described previously [19 ]. The purity of PMN was assessed by preparing cytocentrifuged smears and staining with May-Grünwald-Giemsa (Merck, Darmstadt, Germany) stain. The purity was greater than 96%, and granulocyte viability was >99%, as determined by trypan blue dye exclusion. To investigate the influence of patients’ serum on PMN apoptosis, isolated PMN from healthy volunteers were cultured with 10% serum from patients with ACS, patients with SAP, or control subjects, respectively. To evaluate the influence of platelets on PMN apoptosis, PMN were cocultured with platelets at a ratio of 1:100. Briefly, platelet-rich plasma (PRP) was obtained from heparin anticoagulated blood after centrifugation at 200 g for 10 min. Cell density of 2 x 108 platelets/mL was adjusted by addition of autologous plasma. Then, isolated PMN were resuspended at 2 x 106 cells/mL in RPMI-1640 medium. Aliquots of 500 µL PMN suspension were cultured with 500 µL autologous plasma (control) or 500 µL PRP in the absence or presence of 10 µM ADP for 24 h at 37°C. To examine whether soluble platelet-derived mediators influence PMN apoptosis, aliquots of PRP were stimulated with 10 µM ADP for 15 min at 37°C. After centrifugation at 2000 g for 10 min, the supernatant or the cell-pellet resuspended in autologous plasma was added to PMN culture.

Quantification of PMN apoptosis by PI staining
PMN apoptosis was quantified by flow cytometry as the percentage of cells with hypodiploid DNA [20 ] after 24 h of PMN culture. In each sample, 1 x 104 cells were counted and analyzed using Cell Quest software (Becton Dickinson, San Jose, CA). To remove platelets after the coculture with PMN, samples were incubated for 15 min in ice-cold PBS, supplemented with 2 mM EDTA, and washed twice with hypotonic fluorochrome solution before staining of PMN.

Culture of whole blood
Heparinized blood was diluted 1:5 in RPMI-1640 medium supplemented with 2 mM glutamine, 100 U/mL penicillin G, and 100 µg/mL streptomycin. Then, diluted blood (1 mL) was cultured for the indicated time on 4- or 24-well plates at 37°C in a humidified incubator, under 5% CO2.

Quantification of apoptotic PMN in whole blood
PMN apoptosis in cultured blood was quantified by flow cytometry as the percentage of cells with the decreased CD16 expression on their surface [21 ] after 24 h of blood culture. In each sample, 1 x 104 cells were counted and analyzed using Cell Quest software.

Platelet-activation state
Platelet immunostaining was performed as described previously [22 ]. Fluorescence intensity of 5000 platelets was recorded and analyzed using Cell Quest software.

The evaluation of circulating platelet–PMN conjugates was also performed according to a method described previously [22 ]. In each sample, 1 x 104 cells were counted and analyzed using Cell Quest software. The results were expressed as corrected mean fluorescence value (MFI) calculated as follows: For each sample, the mean fluorescence value for the isotype-matched control was subtracted from the mean fluorescence for the specific antibody.

Assays of TNF-{alpha}, GM-CSF, IL-1ß, and IFN-{gamma}
Serum concentrations of inflammatory cytokines in the study groups were measured using commercially available enzyme-linked immunosorbent assay kits (Biosource International, Germany) according to the manufacturer’s instructions. Detection limits of these assays were 3 pg/mL for TNF-{alpha}, 0.55 pg/mL for GM-CSF, <0.19 pg/mL (ultrasensitive) for IL-1ß, and <2 pg/mL for IFN-{gamma}.

PMN activation assays
As a marker of PMN activation, PMN-derived elastase was determined in patients’ plasma. Elastase coupled to its natural inhibitor {alpha}1-antitrypsin ({alpha}1-AT) was detected as an elastase-{alpha}1-AT complex by an immunoassay procedure (PMN Elastase IMAC, Merck).

CD18 antigen expression was measured in isolated PMN or whole blood by direct immunofluorescence using flow cytometry analysis in the absence (control) or presence of cytokines or ADP for 1 h at 37°C. Aliquots of 100 µL PMN suspension (1x105 cells) or cultured whole blood were incubated with PE-conjugated anti-human CD18 mAb or with isotype-matched control for 30 min at 4°C in the dark. The samples were analyzed by flow cytometry after washing PMN with PBS or erythrocyte lysis in cultured whole blood. PMN were selectively gated using their forward- and side-scatter properties. The results were expressed as MFI.

Statistical analysis
Results are expressed as the mean ± SD. Statistical analysis of the data was performed using the unpaired Student’s t-test; P < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Table 1 shows the baseline characteristics of control subjects and patients with SAP, UAP, and AMI. Patients with ACS had significantly higher levels of troponin I and C-reactive protein as compared with patients with SAP or controls. In addition, patients with AMI showed significantly elevated CK levels.

Delayed PMN apoptosis is a general feature of ACS
The degree of apoptosis of mature human PMN was determined in cells freshly isolated from the circulation or from whole blood cultured for 24 h at 37°C. To assess the loss of DNA content, PI staining, and the CD16 decrease on PMN surface, two well-known markers of apoptosis were measured by flow cytometry. Figure 1 shows a representative original fluorescence histogram from each study group, where PI staining shows apoptosis in isolated PMN, and CD16 decrease indicates PMN apoptosis in cultured whole blood after 24 h. PMN obtained from patients with SAP underwent apoptosis to an extent comparable with PMN from the control group. In contrast, in PMN derived from patients with UAP and AMI, a substantial delay of apoptosis was observed when compared with control PMN or with PMN from SAP patients.



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  		Figure 1. Spontaneous PMN apoptosis in healthy controls and patients with SAP, UAP, and AMI. PMN apoptosis was quantified by flow cytometry: PI staining for loss of DNA content in isolated PMN and decrease of CD16 expression in whole blood PMN. Original recordings of fluorescence histograms of PMN cultured for 24 h. Numbers indicate apoptotic PMN as percent of total cell number. Results are representative of 15 patients or controls in each study group.

 
These results are shown quantitatively in Figure 2 . All patients with AMI (20.3±8.5%) and UAP (28.4±10.1%) showed a significant delay in PMN apoptosis as compared with patients with SAP (44.9±13.6%) or control subjects (51.2±12.6%), as determined by PI staining of isolated PMN (apoptotic cells as percent of total cell number; AMI or UAP vs. SAP or controls, P<0.001; Fig. 2A ). Among patients with ACS, PMN apoptosis was more delayed in AMI than in UAP (P<0.01). No significant difference was seen between patients with SAP and controls (P=ns). The delay in PMN apoptosis in patients with ACS was confirmed in cultured whole blood by measuring the decrease in CD16 expression on PMN (Fig. 2B) . Again, no significant difference was seen between controls (59.8±10.8%) and SAP (52.5±9.5%; P=0.06). However, PMN of patients with UAP (37.4±10%) or AMI (28.2±8.8%) were characterized by a significant delay in apoptosis as compared with SAP or controls (UAP or AMI vs. SAP or controls, P<0.001). Again, PMN of patients with AMI were significantly less apoptotic than PMN from patients with UAP (P=0.005).



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  		Figure 2. PMN apoptosis in healthy controls (C), SAP, UAP, and AMI measured (A) in isolated PMN stained for PI and (B) in whole blood PMN stained for CD16 decrease after 24 h of culture. Circles represent individual measurements and the dotted line, the mean value of each group. *, P < 0.001, versus SAP or C; +, P < 0.01, versus UAP.

 
Serum of ACS patients inhibits apoptosis of PMN from healthy controls
To investigate whether serum components of patients with ACS caused the observed delay in PMN apoptosis, the isolated PMN from healthy volunteers were cultured for 24 h with 20% serum from patients with SAP, UAP, AMI, and from controls (Fig. 3 ). Serum of ACS patients significantly inhibited PMN apoptosis as compared with the serum of patients with SAP or controls (AMI, 30.1±15%; UAP, 32.3±13.5%; SAP, 44.2±17.7%; C, 55.8±16.1%; AMI or UAP vs. C, P<0.0003; AMI or UAP vs. SAP, P<0.05; AMI vs. UAP, P=ns; SAP vs. C, P=ns).



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  		Figure 3. Effect of serum from controls (C), patients with SAP, UAP, and AMI on apoptosis of isolated PMN from healthy volunteers after 24 h of culture. Results are expressed as mean ± SD percentages of PMN apoptosis. *, P < 0.0003, AMI or UAP versus C; +, P < 0.05, AMI or UAP versus SAP; P = ns, AMI versus UAP and SAP versus C (PI staining; n=two separate experiments with 15 serum samples of each study group).

 
Levels of TNF-{alpha}, IFN-{gamma}, GM-CSF, and IL-1ß are increased in the serum of patients with ACS
To prove the hypothesis that the observed delay of PMN apoptosis in patients with ACS depends on serum inhibitors of apoptosis, we determined the serum levels of TNF-{alpha}, IFN-{gamma}, GM-CSF, or IL-1ß in our study groups. As shown in Table 2 , patients with ACS showed significantly higher levels of TNF-{alpha}, IFN-{gamma}, GM-CSF, or IL-1ß as compared with patients with SAP or controls (P<=0.03). In addition, serum levels of IFN-{gamma} and GM-CSF were significantly higher in patients with AMI as compared with patients with UAP (P<=0.02), whereas TNF-{alpha} and IL-1ß were equally elevated in UAP and AMI. Furthermore, serum elevation of TNF-{alpha} and IL-1ß was already detectable in patients with SAP as compared with controls (P<0.05).


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  		Table 2. Serum levels (in pg/mL) of TNF-{alpha}, IFN-{gamma}, GM-CSF, and IL-1ß in the Serum of Controls and Patients with SAP, UAP, and AMI

 
In an additional experiment, we measured the serum levels of high-sensitivity C-reactive protein (hs-CRP) in patients and controls. However, the statistical analysis showed no significant correlation between hs-CRP and CD16 or PI, respectively (data not shown).

Apoptosis of PMN from healthy controls is delayed by IFN-{gamma}, GM-CSF, and IL-1ß but not by TNF-{alpha}
Next, we investigated whether TNF-{alpha}, IFN-{gamma}, GM-CSF, or IL-1ß modulates PMN apoptosis in vitro. Compared with spontaneous apoptosis, IFN-{gamma} (300 U/mL), GM-CSF, and IL-1ß (each at 20 ng/mL) were equally potent in inhibition of apoptosis in isolated PMN after 24 h of incubation (PI staining: control, 52.3±14.2%; IFN-{gamma}, 24.9±12.4%; GM-CSF, 26.9±15.6%; and IL-1ß, 27.6±10.2%; P<0.05 vs. control; n=5). In contrast, TNF-{alpha} (300 U/mL) did not significantly modify apoptosis of isolated PMN (44.7±16.7%; P=ns vs. control) after 24 h of incubation. These results were confirmed in cultured whole blood by measuring the decrease of CD16 on PMN. Again, IFN-{gamma}, GM-CSF, and IL-1ß significantly inhibited PMN apoptosis, whereas TNF-{alpha} did not show any effect (control, 53.9±7.1%; IFN-{gamma}, 20.1±10%; GM-CSF, 29.7±8.2%; and IL-1ß, 26.8±8.4%; P<0.001 vs. control; TNF-{alpha}, 45.5±18.1%; P=ns vs. control; n=5).

Delay of PMN apoptosis by activated platelets can be reversed by aspirin
Besides cytokines, activated platelets are known to modulate PMN functions [23 ]. Particularly in the setting of ACS, platelets become activated, as demonstrated by an increased expression of P-selectin (MFI of CD62P: controls, 9.4±2.6; SAP, 16.4±5; UAP, 26.9±11.1; and AMI, 26.3±8.4; C vs. SAP, UAP, or AMI, P<0.001; SAP vs. UAP or AMI, P<0.01; UAP vs. AMI, P=ns; n=15 for each group of patients and controls). The activation of platelets results in enhanced interactions with PMN, as shown by increased numbers of platelet–PMN aggregates in ACS (MFI of CD61 on platelets adhering to PMN: controls, 19.2±7.4; SAP, 21.3±10.7; UAP, 39.4±8.9; AMI, 42.1±11.7; UAP or AMI vs. SAP or C, P<0.02; AMI vs. UAP, P=ns; C vs. SAP, P=ns; n=15 for each group of patients and controls).

To investigate whether activated platelets may affect PMN apoptosis, isolated PMN from healthy volunteers were coincubated with platelets (Fig. 4A ). First, PMN alone treated with ADP (10 µM) did not show any changes in the degree of apoptosis (control PMN, 49.2±14.2%; PMN with ADP, 42.8±15.4%; P=ns). Second, coculture of isolated PMN with platelets induced a significant delay of PMN apoptosis (34.3±12.3%; P<0.01 vs. control), indicating a partial activation of platelets under culture conditions. Stimulation of platelets with ADP further significantly delayed PMN apoptosis in coculture (21.2±9.7%; P<0.01 vs. coculture without ADP). The influence of platelet activation on the delay of PMN apoptosis was confirmed in cultured whole blood from healthy volunteers (control, 51.6±8.5%, vs. whole blood stimulated with ADP, 27.3±11.3%; P<0.001; Fig. 4C ).



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  		Figure 4. Effect of platelet activation on PMN apoptosis. (A) Flow cytometric analysis of apoptosis (PI staining) in PMN from healthy volunteers, cultured for 24 h in medium alone (Control), with ADP (10 µM), in coculture with unstimulated (ADP–) or with ADP-stimulated platelets (ADP+), n = five to eight, and after pretreatment with aspirin, n = five. (B) Apoptosis of PMN incubated with the cell-pellet or the supernatant of ADP-stimulated platelets. (C) PMN apoptosis in whole blood from healthy volunteers, estimated by the decrease of CD16 after 24 h culture with ADP (10 µM) or after pretreatment of whole blood with aspirin, n = 7. Results are expressed as mean ± SD percentages of PMN apoptosis. (A and B) *, P < 0.05, versus control PMN; §, P < 0.001, versus unstimulated coculture; {dagger}, P < 0.03, versus ADP-stimulated coculture; ADP-stimulated coculture versus supernatant, P = ns. (C) *, P < 0.05, versus untreated whole blood; §, P < 0.04, versus ADP-stimulated whole blood.

 
Furthermore, we investigated whether membrane-bound or soluble factors of activated platelets mediate the observed delay in PMN apoptosis. As shown in Figure 4B , the cell-pellet of degranulated platelets did not significantly delay PMN apoptosis (39.8±12.1%; P=ns vs. control), whereas the supernatant of ADP-activated platelets strongly prolonged the PMN lifespan (27.2±11.7%; P<0.02 vs. control) to a similar extent as found in the ADP-stimulated cocultures (P=ns).

Platelet-induced delay of PMN apoptosis was partially reversed by aspirin, a broadly used antiplatelet medication for patients with CAD (see Table 1 ). Aspirin itself did not influence PMN apoptosis in isolated PMN cocultured with platelets (control, 49.2±14.2%, vs. aspirin 44.2±11.8%; P=ns; Fig. 4A ). Pretreatment of platelets with aspirin before stimulation with ADP reversed platelet-induced delay of PMN apoptosis (30.7±10.5%; P=0.04 vs. ADP). However, the observed effect of aspirin was not a result of a reduction in the number of PMN–platelet aggregates as measured by CD61 expression of platelets adhering to PMN (control, 22.7±15.6; ADP, 319.1±158.5; aspirin, 25.2±17.2; aspirin plus ADP, 311.8±105.1; ADP vs. aspirin plus ADP, P=ns; n=5; data not shown). The inhibitory effect of aspirin on platelet-dependent PMN apoptosis delay was confirmed in whole blood. Although whole blood plus aspirin showed no significant effect on PMN apoptosis (42.1±12.7%; P=ns vs. control; Fig. 4C ), the pretreatment of whole blood with aspirin before stimulation with ADP partially reversed ADP-induced delay of PMN apoptosis (35.1±13.6%; P=0.04 vs. ADP).

Delay of PMN apoptosis is accompanied by PMN activation
The activation status of PMN from patients with ACS was determined by measuring plasma elastase, a proteolytic enzyme released by PMN. PMN-derived elastase was significantly elevated in patients with SAP as compared with controls but still significantly lower as compared with patients with ACS (control, 22.7±11.8; SAP, 36.5±14.8; UAP, 52.6±19; AMI, 64±28.5 ng/mL; control vs. SAP, P<0.004; AMI or UAP vs. control, P<0.0001; AMI or UAP vs. SAP, P<0.02; AMI vs. UAP, P=ns; Fig. 5 ).



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  		Figure 5. Plasma levels of PMN-derived elastase (ng/mL). *, P < 0.0001, versus C; §, P < 0.004, versus C; +, P < 0.02, versus SAP; P = ns AMI versus UAP; n = 15 for each group of patients and controls.

 
To evaluate whether cytokine-induced delay of PMN apoptosis in vitro is accompanied by the activation of PMN, we measured expression of CD18 on isolated PMN after stimulation with cytokines for 1 h at 37°C. TNF-{alpha} (300 U/mL) and to a lesser extent, IFN-{gamma} (300 U/mL), GM-CSF, and IL-1ß (each at 20 ng/mL) significantly up-regulated CD18 expression on isolated PMN, whereas no activation was found in ADP-stimulated (10 µM) PMN (control, 42.1±23.2; TNF-{alpha}, 172.7±56.9; IFN-{gamma}, 70.1±25.2; GM-CSF, 73.6±33.2; IL-1ß, 85.5±37.4; ADP, 44.8±26; control vs. all cytokines, P<0.05; control vs. ADP, P=ns; n=5). In whole blood, all of the cytokines, but also ADP, significantly induced PMN activation as measured by CD18 up-regulation (control, 53.2±15.9; TNF-{alpha}, 136.8±52; IFN-{gamma}, 83.5±14.5; GM-CSF, 97.3±28.3; IL-1ß, 89.7±13.6; ADP, 73.8±24.3; all cytokines or ADP vs. control, P<0.03; n=five). The stimulatory effect of ADP in whole blood, in contrast to its lack of effect on isolated PMN, reflects the influence of platelet activation on PMN functions. In agreement with these results, platelet-dependent activation of PMN by ADP was partially reversible by aspirin when the whole blood was pretreated with 50 µM aspirin for 30 min and subsequently stimulated with ADP (aspirin, 51.9±18.8; aspirin plus ADP, 64.1±23.6; P=ns; n=5).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Atherosclerosis and its complications are accompanied by inflammatory and hemostatic alterations. In ACS, as the final stage of atherosclerosis, PMN are an essential part of an inflammatory process leading to vessel and tissue damage as well as thrombosis. So far, only few studies concentrated on functional characteristics of PMN in ACS [3 , 24 ], and none of them evaluated PMN apoptosis, although induction or prevention of PMN apoptosis is currently discussed as a key event in the control of inflammation [15 ]. The present study demonstrated for the first time a significant delay of PMN apoptosis in patients with ACS as compared with patients with SAP or controls. The delay of PMN apoptosis was even more pronounced in AMI than in UAP and observed in isolated PMN as well as in whole blood. In an in vitro model, serum from patients with ACS strongly delayed apoptosis of normal PMN, whereas serum from patients with SAP or controls did not. Serum analysis of cytokines revealed enhanced levels of TNF-{alpha}, IFN-{gamma}, GM-CSF, and IL-1ß in patients with ACS. Three of these cytokines, IFN-{gamma}, GM-CSF, and IL-1ß, potently induced delay of PMN apoptosis in vitro. Apart from cytokines, the observed delay of PMN apoptosis in ACS may be a result of release of soluble mediators from activated platelets, as demonstrated in reconstructed in vitro experiments.

The delay of PMN apoptosis observed in ACS patients indicates a remarkable shift toward a highly up-regulated inflammatory response, mediated by PMN, with the enhanced risk of tissue damage in these patients. This effect was confirmed in our study, as patients with AMI showed a significantly prolonged PMN lifespan as compared with patients with UAP. When considered on the pathophysiological cellular and molecular level, significant delay of PMN apoptosis in ACS will result in functional longevity with a higher potential for procoagulant activity, leukocyte-plugging in the capillaries, endothelial dysfunction, release of proinflammatory cytokines, and ROS production. All of these aspects were shown to be determinants of tissue damage or clinical outcome in ACS [4 ].

In general, soluble mediators or direct cell-to-cell contact with platelets or endothelial cells can modulate PMN functions. Among the factors specifically preventing PMN apoptosis are IL-6, IL-8, transforming growth factor (TGF)-ß1, and hypoxia, which have been found to be up-regulated in ACS [25 26 27 28 ]. Our study stresses particularly IFN-{gamma}, GM-CSF, and IL-1ß as potent, soluble antiapoptotic mediators for PMN in the serum of patients with ACS. They are derived from activated vascular cells, macrophages, T cells, and PMN themselves and reflect inflammation and atherosclerosis progression [29 ]. Apart from their antiapoptotic effect on PMN, these cytokines influence diverse PMN kinetics and functions such as activation, priming, migration, and cell number [30 ]. Moreover, an elevated serum level of GM-CSF was shown to induce parallel activation of PMN, endothelial cells, and coagulation cascade, all phenomena being an integral part of ACS [31 ].

Our in vitro experiments confirmed the potent antiapoptotic efficacy of IFN-{gamma}, GM-CSF, and IL-1ß. In contrast to these cytokines, TNF-{alpha} follows a biphasic curve with regard to its effect on PMN apoptosis: In our experiments, TNF-{alpha} significantly induced PMN apoptosis within the range of 5–8 h, whereas after prolonged culture (>30 h), its effect was at the level comparable with the apoptosis in control PMN [32 ].

Apart from PMN, activation of monocytes, T cells, and platelets has been reported in ACS. Platelets, in particular, interact with PMN, and both of these cell types can reciprocally regulate their own recruitment at the sites of inflammation and thrombosis. As shown in our study, the prerequisite for PMN–platelet interaction was an activation of platelets, which resulted in enhanced numbers of PMN–platelet aggregates in patients with ACS. Thereby, platelets modulate several PMN functions and activate PMN [33 ]. According to our data, soluble mediators released by activated platelets led to significantly prolonged PMN survival. The specific mediators or mechanisms mediating platelet-induced delay of PMN apoptosis are not completely known. Recently, Brunetti et al. [27 ] found that the platelet-released mediator TGF-ß1 potently inhibited PMN apoptosis, whereas IL-1{alpha} or IL-1ß did not exert any antiapoptotic effect [34 ]. Further research should help to clarify the possible mechanisms and other mediators of apoptosis inhibition.

Enhanced leukocyte-platelet interactions in ACS result in enhanced cardiac tissue damage [35 ]. Consequently, therapeutical intervention with consecutive reduction of leukocyte-platelet interactions (i.e., by GP IIb/IIIa blockade) can protect myocardium [36 ]. In our study, pretreatment with aspirin reversed platelet-induced delay of PMN apoptosis, although the numbers of PMN–platelet aggregates were unchanged in vitro. This finding is in accordance with previous data, where aspirin did not significantly prevent ADP-induced platelet activation and enhanced PMN–platelet interactions. Therefore, specific, direct interference of aspirin with PMN apoptosis and/or functions should be assumed.

PMN apoptosis is associated with changes in PMN functions such as PMN activation. In our study, a marked delay of PMN apoptosis in patients with ACS was accompanied by systemic elevation of PMN-derived elastase, a marker for PMN activation. In vitro, the significant activation of isolated PMN as well as PMN from the whole blood was achieved after supplementation with TNF-{alpha}, IFN-{gamma}, GM-CSF, and IL-1ß. Furthermore, ADP stimulation of platelets resulted in the activation of PMN in whole blood but not in isolated PMN.

Our data show that PMN activation is not associated with delay of PMN apoptosis, as activation of PMN with TNF-{alpha} showed no significant effect on delay of PMN apoptosis in vitro. This was confirmed in our patients with SAP, where marked elevation of serum elastase [37 ] and TNF-{alpha}, but unchanged levels of PMN apoptosis, were observed.

It must also be noted that cytokines and platelet-derived factors are very important but most likely not the only factors influencing PMN apoptosis in the complex pathophysiology of ACS. The limitations of this study were therefore the lack of identification of other mediators of PMN apoptosis, as well as the lack of explanation of the possible cellular mechanisms leading to apoptosis.

In conclusion, the present study demonstrated that apoptosis of PMN from patients with ACS is delayed by proinflammatory mediators such as IFN-{gamma}, GM-CSF, and IL-1ß. The inhibition of PMN apoptosis by these mediators does not only increase lifespan of PMN but also prolongs their functional longevity assessed by a number of parameters including secretion of toxic and prothrombotic products such as PMN-derived elastase. Thus, understanding the mechanisms of PMN apoptosis and its pathophysiologic significance for local and systemic inflammation in vivo may help to design more efficient treatment for patients with ACS.


   FOOTNOTES
 
1 These authors contributed equally to this work. Back

Received July 31, 2003; revised December 3, 2003; accepted December 15, 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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