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To determine changes in leukocyte counts and phagocytic activity of peripheral blood mononuclear (MN) and polymorphonuclear (PMN) cells as potential cellular markers of systemic immunological events in acute myocardial infarction (AMI).
Thirty patients with a first AMI and 30 healthy volunteers were examined. Immunological analyses were performed at admission and repeated at one and seven days after the acute event. MN and PMN cells were obtained from heparinized whole blood after centrifugation and separation on a density gradient, and incubated with a fixed number of heat-inactivated and labelled yeast particles. Total leukocyte counts, leukocyte populations and some parameters of phagocytic activity were determined: percentage phagocytosis, phagocytic index, absolute phagocytic index, count of phagocytes in a fixed volume of peripheral blood (CP) and phagocytic capacity.
Patients with AMI had increased total leukocyte counts accompanied by increased PMN counts, while there were no significant differences in total MN count and MN populations. Except for the phagocytic index, all phagocytic parameters of MN and PMN cells were increased in patients with AMI at admission and on the first day of disease. On the seventh day after AMI only the CP of MN cells had increased significantly in patients with AMI, while percentage phagocytosis, CP and capacity of phagocytosis of PMN cells increased during the acute phase of AMI.
These data suggest that AMI was followed with a strongly systemic inflammatory response to myocardial damage. Furthermore, activated MN and PMN cells may be a significant source of free radicals that may be involved in lipid peroxidation and produce tissue damage in the early postinfarction period.
Ischemic heart disease, and especially acute myocardial infarction (AMI), is one of the most dramatic manifestations in one of the most investigated fields in the past few decades. The beginning and development of both depend on many processes and control mechanisms that occur in the circulation and arterial wall. Acute occlusion of the coronary artery may produce regional ischemia and infarction. This coronary occlusion is mainly the result of thrombosis at the site of injured arterial intima or vasospasm.
Certain populations of peripheral blood leukocytes may play a crucial part in the development of atherosclerotic lession (1,2). Such monocyte/macrophage cells from the circulation migrate into arterial intima where they take part in the elimination of excessive lipoproteins, becoming foam cells (3). There are numerous interactions between monocyte/macrophage cells and other cell types in the arterial wall: endothelium, myocytes, fibrocytes, T lymphocytes, etc. Monocyte/macrophage cells are a very important source of many bioactive molecules such as a cytokines (tumour necrosis factor [TNF], interleukin [IL]-1, IL-6, IL-8, interferon-gamma, etc), enzymes and oxygen free radicals, which destabilize atherosclerotic plaques (4–7). Similar polymorphonuclear (PMN) cells contribute to the formation of atherosclerotic lesions. That is, PMN cells adhere to activated endothelium (8,9), where they may take part in damaging the endothelium by secreting numerous enzymes (collagenases, elastases, etc), cytokines and oxygen free radicals (10) and thus facilitate migration of different cell types and molecules from the circulation into the arterial wall.
During AMI peripheral blood monocyte/macrophage and PMN cells are very active participants in immunological events, seen as an expression of numerous molecules on their surface such as β2 integrins and certain of their very late antigens (11). Both cell types are phagocytic, a process that consists of adherence, diapedesis, chemotaxis, recognition, ingestion, degranulation and oxygen burst. Necrotic tissue is a strong stimulus for macrophages in the ischemic area, whereas antigen-presenting cells take part in phagocytosing and processing necrotic cells and molecules (12), thus initiating numerous intercellular communications. Macrophages may produce TNF in low concentrations which is responsible for expression of adhesion molecules on endothelium, facilitating the adherence of circulating PMN and monocyte cells to endothelium (1,13). IL-1 has a similar effect on endothelium, and is produced by macrophages, which also stimulate endothelial cells to produce other chemokines (14). A strong signal for the migration of peripheral blood monocytes in to an ischemic area is monocyte chemoattractant protein-1 (MCP-1), produced by cardiac myocytes and endothelium in the ischemic area (15). Macrophage-produced IL-12 acts as a differentiation factor of CD4+ T lymphocytes, forming helper T lymphocyte-like cells that produce interferon-gamma, a strong stimulator of phagocytosis (1). Activated CD4+ T lymphocytes produce lymphotoxin, one of the strongest activators of neutrophils (1), but which also acts on the endothelium, increasing adhesivity, production of cytokines (1) and morphological changes significant for diapedesis. T lymphocytes also produce IL-8, which increases adhesivity of the endothelium (1).
Studies of these investigative fields have mostly addressed only particular phases of phagocytosis rather than the complete process. Data from such studies have often been conflicting and incomplete. Therefore, the aim of our investigation was to evaluate the cellular reaction of peripheral blood leukocytes and especially phagocytic activity of mononuclear (MN) and PMN cells in response to the disturbance of homeostasis during AMI.
The study group consisted of 30 patients admitted to the cardiology intensive care unit during autumn and winter 1997. They had a typical history, electrocardiographic manifestations and positive biohumoral syndrome of myocyte necrosis proved by high serum activity of creatine kinase (CK), lactate dehydrogenase, alpha-hydroxybutyrate dehydrogenase and aspartate aminotransferase as a manifestation of AMI. There were 25 men and five women with a mean age of 57.12 years (range 33 to 81). All patients were hospitalized within 4 h of first experiencing pain and all had significant ST elevation on their electrocardiograms at admission. All study patients also had transmural myocardial infarctions (Q wave myocardial infarction). Inferior AMI was diagnosed in 13 patients and anterior AMI in 17 patients. Patients with AMI were treated with vasodilators, analgesics, anticoagulant and antiaggregational therapy without thrombolytic therapy or percutaneous transluminal coronary angioplasty. There were no cases of cardiogenic shock, lethal outcome or spontaneous reperfusion in the patient population during the first week after AMI. All blood samples were obtained three times: immediately after hospital admission and during the morning of the first and the seventh hospitalization days.
Members of the control group were 30 healthy volunteers: 15 men and 15 women with a mean age of 48.31 years (range 27 to 61) without ischemic heart disease demonstrated by normal exercise electrocardiogram and negative biohumoral syndrome of myocyte necrosis. Exclusion criteria were positive parameters of systemic inflammation (high erythrocyte sedimentation rate, high serum fibrinogen concentration) due to other etiologies or positive anamnestic data for other illnesses (eg, autoimmune diseases, acute and chronic infections, systemic and local inflammation, etc) that may have influenced the investigated parameters. None of the AMI patients or control subjects were taking any medications during the week before the investigation. The study was approved by the local ethics committee. Written informed consent was obtained from all subjects.
Total peripheral blood leukocyte counts were determined in a Neubauer hemocytometer (Fein-Optik, Germany) using the original Türck solution. The Türck solution destroys erythrocytes and leukocyte cytoplasm, leaving only leukocyte nuclei, which can be counted. The Neubauer hemocytometer was also used for a relative count (percentage) of peripheral blood MN and PMN cells. Total leukocyte counts and percentage of MN and PMN cells were used to mathematically determine total peripheral blood MN and PMN cell counts.
For peripheral blood lymphocyte and monocyte counts, the method of Horwitz et al (16) was used. This method identifies human MN leukocyte populations by esterase staining. Most peripheral blood monocytes gave a granular and diffuse cytoplasmic esterase reaction product and were phagocytic. The great majority of peripheral blood lymphocytes were found to have discrete granules of reaction product. Percentages of lymphocytes and monocytes in the MN cell population determined by the esterase staining method and total peripheral blood MN cell count were used to determine the lymphocyte and monocyte counts.
MN and PMN cells were isolated from heparinized venous blood using density gradient separation (Lymphoprep 1.077, Nycomed Pharma AS, Norway). Both cell populations were washed three times in Haemacel medium (Jugoremedija, Yugoslavia) and resuspended in complete Haemacel medium, which contained 100 U/mL penicillin (ICN Galenika, Yugoslavia) and 100 mg/L streptomycin (ICN Galenika). Viability remained greater than 95% by acridine orange/ethidium bromide exclusion.
An assay developed by Vujanovic and Arsenijevic (17) was used with minor modification. Isolated MN and PMN cells were suspended in Haemacel medium at a concentration of 1×106/mL in a volume of 400 mL. Heat-inactivated yeast particles labelled with neutral red (Merck, Germany) were than added at a 1:12 effector to target ratio, and cells were centrifuged at room temperature for 5 min at 50 g. The mixed suspension was incubated for 1 h at 37°C in a water bath. Noningested yeast particles were removed by washing twice with ice-cold 0.02% EDTA and once with room temperature Haemacel. The following parameters of phagocytic activity were determined: phagocytic index (PI), defined as the number of yeast particles ingested/cell; percentage phagocytosis (PP), defined as the percentage of cells that ingested at least one yeast particle; absolute phagocytic index (API), the number of yeast particles ingested/100 cells (API=PP×PI); the number of phagocytes (MN or PMN cells) in 1 mL of peripheral blood (CP); and the number of yeast particles ingested by phagocytes in 1 mL peripheral blood, or the phagocytic capacity (CaP).
The extent of myocardial infarction was estimated by calculating the area under the curve of CK, which was measured every 6 h during the first week after AMI. For determination of serum CK, a modified spectrophotometric method of Szasz et al (18) was used.
All values are expressed as mean ± SEM. Statistical evaluation was performed by Student’s t test for paired observation, and one-factorial and two-factorial analysis of variance. Linear regression analysis was performed to evaluate the relation between serum CK concentration and total counts of leukocytes and leukocyte populations, as well as the parameters of phagocytic activity of MN and PMN cells. For all comparisons P<0.05 and P<0.01 were regarded as statistically significant and highly statistically significant, respectively.
In patients with AMI, total leukocyte count was increased (admission day, 15.16±4.62×109/L versus 8.01±1.93×109/L in controls, P<0.01; first day after AMI, 12.89±4.31×109/L versus 8.01±1.93×109/L, P<0.01; seventh day, 12.1±3.67×109/L versus 8.01±1.93×109/L, P<0.01; Figure 1), accompanied by an increase in PMN cell count (admission day, 11.42±4.17×109/L versus 4.85±1.44×109/L, P<0.01; first day, 9.67±3.82×109/L versus 4.85±1.44×109/L, P<0.01; seventh day 8.14±2.97×109/L versus 4.85±1.44×109/L, P<0.01; Figure 1). The total MN cell count was not significantly different between patients with AMI and the control group (admission day, 3.74±1.03×109/L versus 3.16±0.85×109/L, P>0.05; first day, 3.22±1.17×109/L versus 3.16±0.85×109/L, P>0.05; seventh day 3.87± 1.13×109/L versus 3.16±0.85×109/L, P>0.05; Figure 1). There were no differences between the AMI and control groups in counts of MN cells populations: lymphocytes (admission day, 2.60±0.94×109/L versus 2.49±0.69×109/L; first day, 1.99±0.81×109/L versus 2.49±0.69×109/L; seventh day, 2.60±0.89×109/L versus 2.49±0.69×109/L, P>0.05; Table 1) and monocytes (admission day, 1.12±0.47×109/L versus 0.75±0.31×109/L; first day, 1.23±0.58×109/L versus 0.75±0.31×109/L; seventh day 1.27±0.59×109/L versus 0.75±0.31×109/L, P>0.05; Table 1).
Phagocytic activity of peripheral blood MN cells was examined at the same time in 30 patients with AMI. Comparison of patients with their corresponding normal controls showed significant differences in the tested parameters. PP, API, CP and CaP (Table 2) were increased significantly in patients with AMI, whereas PI did not differ (admission day, 3.04±0.37 versus 3.12±0.41, P>0.05; first day, 3.04±0.33 versus 3.12±0.33, P>0.05; seventh day, 3.06±0.28 versus 3.12±0.33, P>0.05; Table 2). CP and API continued to increase during the investigation, whereas PP and CaP started to decrease after the first day of hospitalization, until by the seventh day of AMI there were no significant differences between patients with AMI and the control group. Thus, in the AMI group, more MN cells took part in phagocytosis of necrotic tissue than in the control group, but there was no difference in the capacity of particular cells for phagocytosis.
Changes in the population of PMN cells were similar. Comparison of AMI patients with the control group showed a significant increase in PP and API relative to healthy volunteers (Figure 2). There were similar changes in CP and CaP (Figure 3), which also increased significantly in patients with AMI. These changes lasted from the admission day through the first day and the seventh day, except for API, which started to decrease after the first day of AMI, and by the seventh day of AMI there was no significant difference in this parameter between the groups. Only PI did not show any difference (admission day, 2.51±0.68 versus 2.59±0.39, P>0.05; first day, 2.65±0.42 versus 2.59±0.39, P>0.05; seventh day, 2.50±0.33 versus 2.59±0.33, P>0.05; Figure 4). It was concluded that the intensity of the systemic inflammatory response of PMN cells slowly decreased.
There were only positive correlations between serum CK concentration and total leukocyte count on admission day (r=0.36, P=0.006; Table 3) and the first day (r=0.43, P=0.013; Table 3) as well as total PMN cell count at admission (r=0.49, P=0.009; Table 3) and the first day (r=0.48, P=0.005; Table 3), while there was no positive correlation between serum CK concentration and MN cell count during test days (Table 3). There was also a positive correlation between CK concentration and MN and PMN cell phagocytic activity, but only on the first day after AMI (Tables 4 and and55).
AMI is characterized by many cellular and humoral phenomena during the first days of disease. This study showed the systemic response of peripheral blood leukocytes and particular populations of leukocytes as well as their activity from the perspective of phagocytosis. Our study confirmed the results of several investigations who found that AMI was followed by leukocytosis that was maintained during the acute stage of disease. This phenomenon may be explained by the increased production and release of such cytokines as IL-1, TNF-α, IL-6 and IL-8, known as potent endogenous activators of the acute phase of inflammation. We also found a positive correlation between total peripheral blood leukocyte count and CK concentration as a manifestation of the extent of myocardial infarction at admission and the first day of hospitalization, which was an immune response to the size of tissue injury. But the importance of leukocytosis in AMI is not yet completely clear. For some investigators (19), leukocytosis is one of the crucial parameters for the correct diagnosis of AMI but only in correlation with increased serum activity of CK-MB isoenzyme, and the prognostic assessment of the role of leukocytosis can only be complementary to the role of the other clinical and laboratory parameters. Furman et al (20) noted that peripheral white blood cell count directly correlated with short term hospital mortality and that this association was independent of other prognostic factors, including the extent of AMI. Sanjuan et al (21) confirmed that leukocytosis was only one of the biochemical variables associated with higher AMI mortality. Other researchers associated the appearance of leukocytosis with the etiopathogenesis of AMI. That is, leukocytosis in the acute stage of AMI suggested that AMI is caused mostly by coronary thrombosis in the absence of significant stenosis and, conversely, when the peripheral blood leukocyte count is normal AMI occurs as a result of critical stenosis (22).
We found more significant neutrophilia in patients with AMI than in control subjects, while MN cell count (of both lymphocytes and monocytes) was not significantly different. The neutrophilia was correlated with the extent of myocardial infarction at admission and on the first day of hospitalization, while there was no positive correlation between peripheral blood monocyte count and CK concentration on any of the observation days. Neutrophilia may result from the early appearance of E-selectin, which is synthesized de novo in activated endothelium by TNF-α and IL-1 over 1 to 4 h following activation (1,11). These results agree with the study of Kyne et al (23), who also noted neutrophilia at the early stage of AMI but associated it with early development of heart failure. They suggested that neutrophilia is a parameter that may help determine patients at high risk for heart failure. The appearance of neutrophilia was confirmed in the study of Kassirer et al (24). They showed that neutrophilia during the early stage of AMI corresponded to the appearance of the L-selectin populations of PMN cells and that there was positive correlation between the appearance of this population and the extent of myocardial infarction. Neutrophilia was observed in a study of Krasilnikova et al (25), who also observed monocytosis in patients with AMI. Meisel et al (26) also reported that the appearance of peripheral blood monocytosis correlated with the extent of myocardial damage. These results are contradictory to ours, but may be explained by the fact that we investigated monocyte counts at an early stage of AMI when the immune system has not yet reacted to tissue injury with monocytosis. We did not expect the appearance of monocytosis at so early a phase of AMI, which we confirmed in our study.
There are differing reports of the change in lymphocyte counts during AMI. For example, Kuroki et al (27) and Syrjala et al (28) observed decreased T lymphocyte counts at admission and on the first day of AMI, especially in the ratio of CD4 to CD8 T lymphocytes. In contrast, Tsuchihashi et al (29) did not find significant differences in the percentage of T cells, B cells, CD4-positive cells or CD8-positive cells between patients with AMI and control subjects.
Furthermore, the present study showed that phagocytic activity of peripheral blood MN cells was increased in AMI patients at admission and on the first day of AMI. However, it tended to decrease gradually during the rest of the observation period. CK concentration and MN phagocytic activity were positively correlated only during the first day of AMI.
Several studies noted similar trends. Krasilnikova et al (25) found higher adhesivity and phagocytic ability of peripheral blood MN cells only in patients with penetrating myocardial infarction. Most investigators were concerned with isolated phases of MN cell phagocytosis, which provides only indirect confirmation of our results. The known increase in plasma-soluble intercellular adhesion molecule-1 (ICAM-1) (30–32), vascular adhesion molecule-1 (31,32), endothelial-leukocyte adhesion molecule (ELAM-1) (32) and E-selectin (33) may be associated with adhesion of monocytes and the start of transendothelial migration, but there is no strong evidence for this. These findings suggest that adhesion molecules play an important part in the post-rolling process of leukocyte-endothelial cell interaction in AMI (34). It is well known that AMI is followed by increased serum concentrations of TNF-α and IL-6 (35), which correlates with infarct size and IL-1 (36), with consequent effects on monocyte chemotaxis induced by high levels of expression of MCP-1 on the monocyte surface and its interaction with ICAM-1 on endothelium (14). Similar results found in studies of Matsumori et al (15) and Nishiyama et al (37) confirm that a high serum concentration of MCP-1 is an inflammatory mediator of MN cell phagocytosis in the early stage of disease. It may be a critical step in adhesion and diapedesis of monocytes in injured tissue. But, other than these studies, little is known of phagocytosis of peripheral blood MN cells.
We also evaluated the phagocytic activity of peripheral blood PMN cells and we observed a large increase in this activity during the observed days of AMI. There was a positive correlation between PMN cell phagocytic activity and CK concentration only on the first day of AMI. The majority of other studies have obtained similar results. Dimitrijevic et al (38) noted that chemotaxis and metabolic activity of peripheral blood PMN cells were enhanced during AMI but not phagocytosis. PMN cell activation may result from contact with the ligands expressed on endothelial cells or in response to soluble stimuli released from ischemic tissue into the plasma. Most authors have based their investigations on isolated phases of phagocytosis, which may indirectly confirm some of our results. It is known that TNF-α and IL-1 increase adhesivity of the endothelium to human neutrophils by inducing the expression on the endothelium surface of ligands such as ELAM-1 and ICAM-1 (9). On the other hand, increased expression of lymphocyte function is associated with antigen-1 on the surface of PMN cells during the acute phase of AMI, which may contribute to their adhesion to endothelium in ischemic tissue (39). Adhesivity to endothelium may be anhanced by acidosis generated during tissue ischemia by CD-18 mediated PMN cell adhesion (8). PMN cells show an increasing chemotaxis (40) produced by the chemotactic molecules leukotriene B4, complement molecule C5a, intercrines, etc (11). Reisenberg et al (41) detected in sera of all AMI patients a high level of IL-8 and suggested that in addition to its chemotactic role it may be one of the major contributors to the priming of PMN cells in these patients. This priming of PMN cells is contributed by TNF-α, which is also involved in enhancing degranulation of these cells (42). Evidence for the respiratory burst of PMN cells is contradictory. That is, Guarnieri et al (43) noted reduced oxidative activity in circulating PMN cells emphasizing that PMN cells were functionally inhibited immediately after AMI. In contrast, several studies have found increased production and release of free oxygen radicals originating from circulating PMN cells (10,41,44) and suggested their possible role in myocardial reperfusion injury.
Generally, we have shown a systemic response of peripheral blood leukocytes during AMI, reflecting their phagocytic activity. These results show that AMI is followed by numerous changes in the cellular component of the immune system, but many details are not yet completely clear in this field of investigation.
The authors thank the staff of the Laboratory for Clinical and Experimental Immunology of the Clinical Hospital Centre in Kragujevac for their excellent technical assistance.