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Increased expression of interleukin-10 in unstable plaque obtained by directional coronary atherectomy

Kensaku Nishihira, Takuroh Imamura, Atsushi Yamashita, Kinta Hatakeyama, Yoshisato Shibata, Yoshitatsu Nagatomo, Haruhiko Date, Toshihiro Kita, Tanenao Eto, Yujiro Asada
DOI: http://dx.doi.org/10.1093/eurheartj/ehl058 1685-1689 First published online: 13 June 2006

Abstract

Aims The present study investigates the expression and localization of interleukin (IL)-10, an important anti-inflammatory cytokine, in atherectomy specimens from patients with stable and unstable angina.

Methods and results Twenty-two patients with stable angina and 21 with unstable angina who underwent directional coronary atherectomy for de novo lesions were studied. The atherectomy specimens were morphologically assessed and immunohistochemically stained with antibodies for IL-10, macrophages, smooth muscle cells, and endothelial cells. The localization and immunopositive areas were evaluated using an image analysing system. Immunoreactivity for IL-10 was detected in coronary plaques, especially in macrophages. Immunopositive areas of macrophages and IL-10, as well as the incidence of thrombus formation, were significantly greater in specimens from patients with unstable angina than in those from patients with stable angina (macrophages, P<0.001; IL-10, P<0.05; thrombus formation, P<0.05; respectively). Even after adjustment, IL-10 expression and the incidence of thrombus formation were significantly greater in the unstable angina group (P<0.05, each). The immunoreactivities for smooth muscle cells and endothelial cells did not differ between the two groups.

Conclusion IL-10 was more frequently expressed in specimens from patients with unstable angina. This finding might contribute to a better understanding of plaque instability.

  • Atherectomy
  • Atherothrombosis
  • Immunohistochemistry
  • Interleukin-10
  • Unstable angina

Introduction

Inflammation plays a major role in the pathogenesis of atherosclerosis and plaque instability.1,2 We and other investigators showed that a variety of pro-inflammatory cytokines, including interleukin (IL)-1β, IL-6, tumour necrosis factor-α, and C-reactive protein are expressed in human atherosclerotic plaques.13 These cytokines contribute to plaque formation and its instability. Although the roles of pro-inflammatory cytokines in atherosclerosis have been extensively studied, those of anti-inflammatory cytokines have not been well elucidated.

IL-10 is one of the most important anti-inflammatory cytokine that is mainly produced by macrophages and T-helper (Th) type-2 lymphocytes, and inhibits a broad array of immune parameters.4,5 This anti-inflammatory cytokine is also expressed in human atherosclerotic plaques.6 Recent studies in vitro and in vivo have shown a protective role of IL-10 in both atherogenesis and plaque instability.4,5 However, IL-10 localization in coronary plaques and its relationship with clinical types of angina have not been examined. We therefore immunohistochemically investigated the localization of IL-10 in a culprit lesion obtained by directional coronary atherectomy (DCA) in patients with either unstable or stable angina. These findings provide further information on another potential mechanism of coronary plaque instability.

Methods

Patient selection

Between April 2002 and March 2005, we recruited 46 consecutive patients (24 with stable angina and 22 with unstable angina) who had undergone DCA for a de novo lesion. Exclusion criteria were patients with serious infectious diseases, malignancies, or chronic inflammatory diseases. All patients provided written informed consent to participate in the study, and the Institutional Ethics Committees approved the study protocol. Although all patients agreed to enter the study, two patients with malignancies and one with an infectious disease were excluded and were not further examined. Finally, the study cohort comprised 22 patients with stable angina, classes 1–3 according to the Canadian Cardiovascular Society (CCS) classification,7 and 21 with unstable angina, classes I–III according to Braunwald's classification.8 Hypertension, hyperlipidaemia, diabetes mellitus, smoking, obesity, and family history of coronary artery disease (CAD) represented risk factors for CAD. A culprit lesion was identified by a combination of electrocardiographic findings, angiographic lesion morphology, and left ventriculographic or echocardiographic wall motion abnormalities.

Immunohistochemistry and quantitative methods

Specimens obtained at DCA were immediately fixed in 4% paraformaldehyde and embedded in paraffin. Sections were stained with haematoxylin–eosin and serial sections were also examined immunohistochemically using primary antibodies against α-smooth muscle actin (smooth muscle cells, DakoCytomation, Glostrup, Denmark), von Willebrand factor (VWF) (endothelial cells, DakoCytomation), CD68 (macrophages, DakoCytomation), and IL-10 (R & D Systems Inc., Minneapolis, MN, USA). The sections were stained using EnVision+ kits (DakoCytomation). Horseradish peroxidase activity was visualized with 3,3′-diaminobenzidine containing hydrogen peroxide. To identify cell types that stained for IL-10, we also performed immunodouble staining with IL-10 and the above antibodies. IL-10 was visualized using New Fuchsin kit (red; Nichirei, Tokyo, Japan) and other antibodies were visualized using 3,3′,5,5′-tetramethylbenzidine (blue; Vector Laboratories Inc., Burlingame, CA, USA). Immunopositive areas of these antibodies, except for VWF, were quantified using a colour imaging analysis system (MacSCORP, Mitani, Fukui, Japan) and are expressed as the ratio of positively stained areas per total tissue as described.9 The density of VWF is expressed as the number of tubuloluminal VWF-positive capillaries per mm2. Morphometric analyses were performed by two investigators (A.Y., K.H.) who were blinded to the patients' clinical classification.

Statistical analysis

Data are expressed as medians with interquartile ranges (IQRs). Differences between two groups were analysed using an unpaired Student's t-test or the Mann–Whitney U test when the variance was skewed. Categorical variables were compared by Fisher's exact probability test. Statistical comparisons for greater than or equal to three groups were analysed by one-way ANOVA and by post hoc multiple comparisons using Scheffe's test. We also assessed independent predictors of the symptomatic unstable plaque using multiple logistic regression analysis with independent variables, being those with P<0.05 in univariate analyses. All tests were two-sided and a P-value of <0.05 was considered statistically significant.

Results

Table 1 lists the clinical characteristics of the patients. Risk factors for CAD and administered drugs did not significantly differ between patients with stable and unstable angina, expect for the prevalence of smoking which was significantly more common in the latter group. Figure 1 shows representative immunohistochemical staining for macrophages (Figure 1A and C) and IL-10 (Figure 1B and D) in coronary plaques obtained from a patient with stable angina (Figure 1A and B), and those from a patient with unstable angina (Figure 1C and D). Significantly more areas were immunopositive for macrophages and IL-10 in patients with unstable, than with stable angina [23.4% (IQR 10.8–34.7) vs. 9.3% (IQR 3.1–21.6), P<0.001; 7.3% (IQR 1.9–16.9) vs. 3.1% (IQR 0.4–5.9), P=0.041, respectively, Table 2]. Multiple logistic regression analysis also showed that the difference in IL-10 expression between these groups remained statistically significant after the inclusion of all variables that were significant in the univariate analyses listed in Tables 1 and 2 (P=0.038, Table 3).

Figure 1

Representative immunohistochemical staining of DCA specimens from patients with stable (A and B) and with unstable (C and D) angina for macrophages (A and C) and IL-10 (B and D). Immunodouble staining (E) demonstrated that IL-10 immunoreactive cells (red) are mainly macrophages (blue) (indicated by arrow heads).

View this table:
Table 1

Clinical characteristics of study patients

Stable angina (n=22)Unstable angina (n=21)P-value
Age (years),
 Median62560.618
 IQR52–7151–71
Men (%)20 (91)16 (71)0.24
Risk factors
 Hypertension (%)12 (55)17 (81)0.104
 Hyperlipidaemia (%)13 (59)9 (43)0.366
 Diabetes mellitus (%)8 (36)5 (24)0.509
 Smoker (%)9 (41)17 (81)0.012
 Obesity (%)3 (14)6 (29)0.281
 Family history (%)3 (14)5 (24)0.457
Medication (on admission)
 Beta-blockers (%)4 (18)1 (5)0.345
 Nitrates (%)8 (36)9 (43)0.76
 Calcium antagonists (%)10 (45)8 (38)0.76
 Aspirin (%)21 (95)16 (76)0.095
 ACE-I (%)4 (18)3 (14)>0.999
 Statins (%)10 (45)4 (19)0.104
  • Hyperlipidaemia (total cholesterol >220 mg/dL or patient on lipid-lowering therapy); obesity (BMI >30 kg/m2); ACE-I, angiotensin converting enzyme-inhibitors.

  • Values of P<0.05 were considered significant.

View this table:
Table 2

Histological characteristics

Stable angina (n=22)Unstable angina (n=21)P-value
Incidence of thrombus formation8 (36%)16 (73%)0.014
Immunohistochemistry
VWF-positive capillaries (per mm2)0.084
 Median1734
 IQR8–2612–48
CD68 (% positive area)<0.001
 Median9.323.4
 IQR3.1–21.610.8–34.7
Alpha-smooth muscle actin (% positive area)0.382
 Median38.325.1
 IQR21.2–54.720.9–42.5
IL-10 (% positive area)0.041
 Median3.17.3
 IQR0.4–5.91.9–16.9
  • Values of P<0.05 were considered significant.

View this table:
Table 3

Multivariate predictors of histological characteristics with unstable angina

OR95% CIP-value
Incidence of thrombus formation8.141.25–52.970.028
CD681.050.98–1.120.159
IL-101.161.01–1.330.038
  • Variables were those with P<0.05 in univariate analyses (smoking, incidence of thrombus formation, CD68, and IL-10). Values of P<0.05 were considered significant.

Immunodouble staining demonstrated that the cells that were immunoreactive for IL-10 were mainly macrophages (Figure 1E). Therefore, the ratio of IL-10- to CD68-positive areas (IL-10/macrophage ratio) within the matched area was examined to assess whether the expression of IL-10 by macrophages differed between clinical types of angina. The IL-10/macrophage ratio also tended to be higher in unstable, than in stable angina [0.42 (IQR 0.10–0.63) vs. 0.13 (IQR 0.05–0.48), P=0.076].

Moreover, the incidence of thrombus formation was significantly higher in patients with unstable, than with stable angina (73 vs. 36%, P<0.05, Tables 2 and 3). Although unstable plaques tended to contain more VWF-positive capillaries than stable plaques, the difference was not significant [34 (IQR 12–48) vs. 17 (IQR 8–26) per mm2, P=0.084, Table 2] and the immunopositive areas of smooth muscle cells also did not significantly differ. In addition, IL-10 expression on DCA specimens did not significantly differ between smokers and non-smokers [4.5% (IQR 2.1–9.8) vs. 3.0% (IQR 0.4–11.8), P=0.81].

Among 21 patients with unstable angina, IL-10 positive areas tended to differ in accordance with the three categories of Braunwald's classification, but the difference was not significant [class I, 6.5% (IQR 0.4–9.8); class II, 6.7% (IQR 1.1–14.5); class III, 18.1% (IQR 3.3–21.9)].

Discussion

Here, we demonstrated for the first time, that IL-10 is present in coronary culprit plaques obtained by DCA. More areas were immunopositive for IL-10 on DCA specimens in patients with unstable, than with stable angina and the immunopositive cells were mainly macrophages.

Inflammation is a critical process of atherogenesis and plaque instability.1,2 A balance between pro-inflammatory and anti-inflammatory responses is one of major determinants of the onset of acute coronary events. The involvement of pro-inflammatory factors, such as IL-1, IL-6, and C-reactive protein in atheromatous plaques predicts an unfavourable outcome in patients with acute coronary syndromes,13 whereas little is known about the role of anti-inflammatory cytokines. IL-10 is one of the most important mediators that physiologically limits and down-regulates inflammation.4,5 The primary role of IL-10 is to suppress immune function by blocking the synthesis of pro-inflammatory cytokines, such as IL-1, IL-6, interferon-γ, and tumour necrosis factor-α in T-cells and monocytes/macrophages.4,5 It also inhibits many cellular processes that might play important roles in plaque progression, rupture, or thrombosis, including nuclear factor-κB activation, metalloproteinase production, tissue factor expression, and cell death.4,5,10

Recent clinical studies have shown that the serum level of IL-10 is a predictive marker for acute coronary events.11,12 Serum IL-10 levels, in contrast to local tissue levels, were significantly lower in patients with unstable, than with stable angina.11 Elevated IL-10 serum levels diminished the risk of death and recurrence of myocardial infarction.12 Fichtlscherer et al.13 demonstrated that increased IL-10 serum levels were associated with improved systemic endothelial vasoreactivity in patients with elevated C-reactive protein serum levels. These findings suggest that a reduced IL-10 serum level is not only a marker of plaque instability and onset of acute coronary syndromes, but also predictive of a poor prognosis after acute coronary events. Nonetheless, reports indicate that IL-10 is up-regulated in the reperfused myocardium during myocardial ischaemia-reperfusion following acute coronary events.4,14 In addition, inflammatory factors such as C-reactive protein and tumour necrosis factor-α, which are rich in unstable coronary plaques,2,3 also up-regulate IL-10 production.15,16 IL-10 appears to act in a feedback loop to inhibit continued pro-inflammatory cytokine production. Therefore, IL-10 levels in tissues and cells of plaques are probably more relevant than those in the circulation, but such data are unavailable. The present study found that immunoreactive IL-10 was more prominent in coronary plaques from patients with unstable, than with stable angina. We also found that the IL-10/macrophage ratio tended to be higher in unstable, than in stable angina. Several investigators have identified IL-10 in advanced human atherosclerotic lesions, but not found in non-atherosclerotic arteries,6 and the IL-10/macrophage ratio is significantly different in the American Heart Association classification of human atherosclerotic lesions.17 Based on this and our evidence, IL-10 expression in macrophages might vary with pathological conditions. Moreover, recent studies have demonstrated that an IL-10 deficiency enhances early atherosclerotic lesion formation,1820 and that IL-10 supplementation or overexpression reduces the size of lesions.18,19,21 Although we could not determine the physiological role of IL-10 in plaques, these studies suggest that IL-10 plays a protective role in plaque progression and instability.

Macrophage accumulation is one of the characteristic features of vulnerable plaques, and the apoptosis of these cells might promote plaque instability.1,2 Immunoreactive IL-10 was mainly observed in macrophages of coronary plaques. Mallat et al.6 have identified immunoreactive IL-10 mainly in macrophages but also in smooth muscle cells and in the extracellular matrix of advanced human atherosclerotic plaques obtained from carotid arteries and abdominal aortic aneurysms. Although we could not exactly explain the difference between these and our findings, differences in immunohistochemical methods and plaque samples are probably associated. Macrophages stimulated by oxidized low-density lipoprotein (LDL) release many pro-inflammatory cytokines, which contribute to plaque instability,1 and studies in vitro have demonstrated that oxidized LDL also induces IL-10 release from macrophages.22 Halvorsen et al.23 recently documented the anti-apoptotic effect of IL-10 in oxidized LDL-stimulated macrophages from patients with acute coronary syndromes, but not in macrophages from healthy controls. These results indicate one possible mechanism through which IL-10 stabilizes plaques.

In addition to oxidized LDL, thrombus could play a role in inducing IL-10 expression. Platelets activated after exposure to high shear stress and thrombin, the key enzyme in the coagulation pathway and in thrombosis, enhance IL-10 production in human mononuclear leukocytes in vitro.24,25 In addition, IL-10 expression is elevated in the vascular wall during venous thrombosis, and IL-10 neutralization increases such thrombosis in vivo.26 The present study showed that the incidence of thrombus formation and IL-10 expression were significantly higher in patients with unstable, than with stable angina. Our results support these previous findings and indicate that IL-10 is induced to maintain equilibrium in the context of local inflammatory responses at the sites of thrombus formation.

The limitations of this study are as follows. First, the timing of DCA is related to the last anginal episode and the amount of IL-10. However, IL-10 positive areas did not significantly differ among the three categories of Braunwald's classification. Secondly, although this was a cohort study, the small sample size might not have been sufficient to reach a definitive conclusion. Further investigations are required to confirm our observations and to evaluate the effects of drugs such as statins on IL-10 regulation. Thirdly, the relationship between IL-10 levels in serum and tissue could not be assessed as blood samples from the patients were unavailable. Finally, the present study could not determine the physiological role of IL-10 in plaques. Therefore, basic experiments are required to resolve this issue.

In conclusion, we demonstrated for the first time that IL-10 is expressed in coronary culprit lesions. Moreover, more IL-10 was expressed in coronary plaques from patients with unstable, than with stable angina. These findings might contribute to a better understanding of the pathogenesis of plaque instability.

Acknowledgements

This study was supported in part by Grants-in-Aid for Scientific Research (c) (No.14570153) and for the 21st COE Research (Life Science) from the Ministry of Education, Science, Sports and Culture, Japan.

Conflict of interest: none declared.

References

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