levels of serum il-1 , il-2, il-8

Objectives: Inflammation is the most importantmechanism of plaque disruption playing an essentialrole in acute coronary syndromes. It is controversialwhether the inflammatory mediators are the cause orthe result in the development of plaque rupture.Stimulation of interleukins increases adhesion mole-cules, fibrinogen and plasminogen activator inhibi-tors, which cause the activation of inflammation andthrombosis. However, the importance of interleukinsin acute coronary syndromes has not been clearlydefined. We did not find any article concerningrelations between the levels of serum interleukin(IL)-1b, IL-2, IL -8 and tumor necrosis factor (TNF)-a inpatients with unstable angina pectoris (UAP). So theaim of this study was to determine the levels of serumIL-1b, IL-2, IL-8 and TNF-a during the early stage ofUAP.Methods and results: Thirty-seven patients with UAP(12 females and 25 males; mean age, 57.59/9.7 years)within 6 h of admission and 20 healthy volunteers(eight females and 12 males; mean age, 51.39/6.3years) were included in the study. IL-1b, IL-2, IL-8 andTNF-a levels were measured using the enzyme-linkedimmunosorbent assay method. Patients with acute orchronic inflammation, renal failure or chronic heartfailure were excluded from the study. The age, genderand risk factors of the study and control groups weresimilar. The levels of IL-1b, IL-8 and TNF-a weresignificantly increased (p B/0.0001, p B/0.001 andp B/0.016, respectively) in patients with UAP. Therewas no difference of IL-2 levels between the UAPgroup and controls.Conclusion: We detected high levels of IL-1b, IL-8 andTNF-a in patients with UAP during early phase. Wesuggest that proinflammatory cytokines (e.g. IL-1b,IL-8, TNF-a) may play an important role in thedevelopment of atherosclerosis and its complica-tions.

Key words: Unstable angina pectoris, Inflammation, In-terleukins

Mediators of Inflammation, 12(6), 361�/365 (December 2003)

Levels of serum IL-1b, IL-2, IL-8and tumor necrosis factor-a inpatients with unstable anginapectoris

Ali Ozeren1,CA, Mustafa Aydin1, Mehmet Tokac2,

Nejat Demircan3, Murat Unalacak3, Ahmet Gurel4

and Mehmet Yazici2

1Department of Cardiology, 3Department of FamilyMedicine and 4Department of Biochemistry,Karaelmas University, Faculty of Medicine, TipFakultesi, Kardiyoloji Anabilimdali, 67600 Kozlu,Zonguldak, Turkey; 2Department of Cardiology,Faculty of Medicine, Selcuk University, Konya, Turkey

CACorresponding authorTel: �/90 3722610169Fax: �/90 3722610155E-mail: [email protected]

Introduction

Atherosclerosis is currently one of the main causes ofdeath. A great majority of deaths related to athero-sclerosis occur due to acute complications, mainlybased on plaque disruption.1,2 Inflammation is themost important mechanism of plaque disruption ofwhich it is crucial to reveal the molecular mechan-isms.3,4

Previously, the appearance of immune cells at theischemic site of myocardial tissue was believed to bea response to tissue injury. Inflammatory processesare now recognized to play a central role in thepathogenesis of atherosclerosis and its complications.A growing body of evidence suggests that unstableangina is associated with local and systemic activa-

tion of the immune system. Plasma levels of severalinflammation markers have been found to be asso-ciated with future cardiovascular risk in a variety ofclinical settings. These markers include cell adhesionmolecules, cytokines, pro-atherogenic enzymes andC-reactive protein (CRP).5

Circulating markers may consist of cytokinesdirectly released from inflammatory cells present inthe plaques and tissues exposed to recurrent ische-mia as well as other reactants produced in responseto these cytokines such as adhesion molecules andacute phase proteins. Recent studies suggest thatmarkers of inflammation may reflect different aspectsof the atherothrombotic process at different points inthe continuum of acute coronary syndromes (ACS).Inflammatory markers have a potential role for the

Short Communication

ISSN 0962-9351 print/ISSN 1466-1861 online/03/60361-05 – 2003 Taylor & Francis LtdDOI: 10.1080/09629350310001633360

361

prediction of risk for developing coronary arterydisease (CAD), and could correlate with severityand future risk for CAD.6

Inflammation markers such as CRP, fibrinogen andcytokines have been implicated in the developmentand progression of CAD.7�13 However, their role inthe development of ACS has not been clearly defined.We did not find any article concerning the relationsbetween the levels of serum interleukin (IL)-1b, IL-2,IL-8 and tumor necrosis factor (TNF)-a in patientswith unstable angina pectoris (UAP). So the aim ofthis study is to determine the levels of serum IL-1b,IL-2, IL-8 and TNF-a during the early stage of UAP.

Materials and methods

Patients with new onset, Braunwald Class III restingangina within 6 h, but not preceding acute myocar-dial infarction, were considered as having UAP andwere included in the study (12 females and 25 males;mean age, 57.59/9.7 years). The study group did nottake any medicine and had a fairly unremarkablemedical history. Twenty patients (eight females and12 males; mean age, 51.39/6.3 years) with normalcoronary arteries on angiogram were considered thecontrol group. Patients with evidence of any infec-tious disease, significantly higher erythrocyte sedi-mentation rate (�/20 mm/h), high fever (]/38.38C),immunological disorders, neoplastic diseases, chestpain lasting more than 6 h and normal coronaryarteries were not included in the study. Finally, 37patients remained in the UAP group.

All of the patients with UAP were hospitalized andtheir medications were initiated accordingly. Allpatients received acetyl salicylic acid, b-blocker,weight-adjusted low molecular weight heparin andlipid lowering therapy. Thirty-seven patients enrolledin the study underwent coronary angiography. Sig-nificant CAD was defined as more than 50% narrow-ing of the luminal diameter in a major epicardialvessel. Blood samples were obtained from the UAPgroup on admission (within a maximum 6 h after the

onset of pain) before angiographic investigation, toreveal biochemical parameters and interleukin levels.Blood samples were centrifuged at 5000 rpm for 5min and the collected serum samples were stored at�/808C for a maximum time period of 1 month. IL-1b,IL-2, IL-8 and TNF-a kits were purchased from R&D†

(Roche Diagnostics, USA) and Biosource (USA), andthe results were interpreted according to the instruc-tions given by the manufacturer.

All data are presented in the format of the mean9/

standard deviation. Comparisons between twogroups were performed using a parametric one-wayanalysis of variance test. p B/0.05 was consideredstatistically significant.

Results

Age, gender, and risk factors (e.g. smoking, lipidprofiles, hypertension, and diabetes mellitus) of thepatients are presented in Table 1. There was nostatistically significant difference between study andcontrol groups.

In the group of UAP, the levels of IL-1b, IL-8 andTNF-a were significantly increased (p B/0.0001, p B/

0.001, p B/0.016 and p B/0.05, respectively) (Table 2).There was no difference of IL-2 levels between theUAP group and the control group.

Discussion

Recently, the role of inflammation in the pathogen-esis of atherosclerosis has been understood better. Bymeans of acute phase reactants, interleukins increaseboth inflammation and smooth muscle hyperpla-sia.14,15 Interleukins, which are soluble hormone-like protein substances, mediate the functions ofmessage production and interaction between im-mune system cells and determine the immuneresponse.

Stimulation of interleukins increase adhesion mo-lecules, and fibrinogen and plasminogen activator

Table 1. Characteristics of patients

UAP group(n�/37)

Control group(n�/20)

p value

Age (years) 589/10 519/6 NSSex 25 male/

12 female12 male/8 female

NS

Smokers (%) 24(65) 12(60) NSHypertension (%) 16(43) 8(40) NSDiabetes mellitus (%) 9(24) 4(20) NSHeredity (%) 8(21) 3(15) NSTotal cholestrol 2019/40 1969/31 NSLow-density lipoprotein cholestrol 1469/27 1359/26 NSHigh-density lipoprotein cholestrol 329/6 379/6 NSTriglyceride 2129/95 1439/64 NS

NS, Not significantly different.

A. Ozeren et al.

362 Mediators of Inflammation � Vol 12 � 2003

inhibitors cause activation of thrombocytes and thusstimulation of thrombosis.16 Ischemia causes releaseof TNF-a and IL-1b from mononuclear cells. It hasbeen proved that there are elevated levels of inter-leukins in serum of patients with UAP, which carryproinflammatory and procoagulant properties andthe degree of elevation of the interleukin level isrelated to prognosis.17

Interleukin-1b

IL-1b is produced largely by activated macrophages,endothelial cells, and also by vascular smooth musclecells.18,19 IL-1b actively participates in the regulationof vascular cell functions, including the stimulation ofleukocyte adhesion to the endothelial cells, perme-ability of vessels, matrix metalloprotease production,suppression of vascular contractility, regulation of thepathways of coagulation within the cell and theproduction of procoagulator.16 It is clear that itslevels increase during ischemic heart disease. IL-1bcontributes to the development of atherosclerosis.Simon et al .20 stated that there was a significantincrease in IL-1b values in UAP patients comparedwith stable angina patients.

We found statistically significant increase in IL-1blevels of the study group. The effect of IL-1b on acuteprocess was not clear. IL-1b might be responsible forplaque rupture and triggering ACS. However, IL-1bcould be released from the vascular endothelium orischemic myocardium. Since we could measure IL-1bonly at admission, we could not differentiate thesource of this increase. In addition to CRP evaluation,we suggest that IL-1b levels could be used fordetection of high-risk patients.

Interleukin-2

In normal conditions immunologic response does notshow IL-2 within circulation. IL-2 causes angiogen-esis. All IL-2, IL-4, IL-6 and TNF-a can induce releaseof IL-2 and its receptors. IL-2 has a central role in thedevelopment of cell-mediated immunity and also itserves as a key factor in the induction of a complexnetwork of cytokines.21

Mazzone et al .22 showed that IL-2 levels increasedin patients with CAD, but no significant increase was

observed in ACS. Takeshita et al .23 found that serumIL-2 receptors were significantly lower in ACS com-pared with control. Mizia-stec et al . reported thatpatients with CAD, irrespective of the form of thedisease, have higher serum levels of pro-inflamma-tory and anti-inflammatory cytokines than controlsubjects. Increased concentrations of IL-2 in UAP maysuggest additional immunologic activation.24 On thecontrary, levels of IL-2 and IL-2 receptors werereported to be elevated significantly in patients withstable angina, but not in patients with UAP.25

In our study, the UAP group revealed a lower butnot statistically significant level of IL-2 compared withthe control group. This result seems to be inaccordance with the study of Simiti et al .25 Since IL-2 is an anti-inflammatory cytokine, we thought that itmight be downregulated first, and later on reachesnear normal levels in the early stages of ACS. Wemeasured IL-2 levels only at admission, so we cannotestimate the profile of IL-2 levels. The role of IL-2 onACS has not been clearly defined as the results ofprevious studies are contradictory. Further studies arenecessary in this issue.

Interleukin-8

Mononuclear cells, fibroblasts, endothelial cells, ker-atinocytes and thrombocytes synthesize IL-8. Itssynthesis is stimulated by IL-1 and TNF-a. Chemo-tactic features of IL-1 and TNF-a are mediated by IL-8,which is a crucial chemokine known to attract andactivate neutrophils as well as T lymphocytes, and tocontrol their interaction. There are only a few studiesin the literature on the IL-8 levels in patients withmyocardial infarction. Simiti et al . reported that theincreased plasma IL-8 levels within the first 24 h afterthe spontaneous episode of angina could represent amarker of primary UAP, Braunwald’s class III-B. Butthe plasma IL-8 levels do not increase in stableangina.25

Qi et al . demonstrated that plasma IL-8 levelsincrease as a reflector of the abnormal coagulationactivity in patients with UAP after coronary angio-plasty.26 Miya et al . reported that IL-8 seems to havea role in the pathogenesis of acute coronary arterythrombi. This hypothesis leads us to test thoseinterventions on the influence of IL-8 secretion inthe early phase of coronary thrombus formation.27 Inthe literature on the pathologic and biochemicalinvestigations of the atherosclerotic coronary pla-ques, it has been stated that the level of IL-8 increasedas an angiogenic factor.27�29

In our study, the levels of IL-8 in UAP cases werefound significantly increased compared with normalhealthy subjects. Our results are in accordance withliterature clues. We suggest that IL-8 has an importantrole in inflammation and coagulation in UAP cases.We did not conclude any definitive relationship

Table 2. Levels of serum IL-1b, 1L-2, 1L-8 and TNF-a

UAP group(n�/37)

Control group(n�/20)

p value

IL-1b (ng/dl) 3789/34 119/14 B/0.0001IL-2 (ng/dl) 119/14 159/19 NSIL-8 (ng/dl) 4189/506 879/154 B/0.001TNF-a (ng/dl) 1009/201 199/9 0.016

Values are expressed as mean9/standard deviation. NS, Notsignificantly different.

Levels of serum IL and TNF-a in VAP

Mediators of Inflammation � Vol 12 � 2003 363

between other inflammatory parameters and coagu-lation status, since we did not measure them.

Tumor necrosis factor-a

TNF-a plays a role in the processes of coagulation,ischemia and reperfusion injury. TNF-a may have acertain role in the development of ischemia bycausing release of endothelial adhesion molecules,activation of leukocytes and secretion of thrombocyteactivating factors. Endothelial cells are highly sensi-tive to TNF-a. By the effect of TNF-a the productionof adhesion molecules and procoagulants increases,whereas the synthesis of protein-C is suppressed.30

Proving the high serum levels of TNF-a followingacute myocardial infarction observed in animalmodels, TNF-a has been suspected to be relatedwith acute myocardial infarction pathogenesis.30

Koukkunen et al . reported that a 3.5-fold increasewas observed in fibrinogen and TNF-a. They sug-gested two underlying sources of events; the first isthe ‘inflammation’ factor that includes CRP, fibrino-gen and IL-6, and the second is the ‘injury’ factor thatincludes troponin-T, creatine kinase-MB mass andTNF-a. Both of these factors were independentpredictors of the risk of coronary death and othermajor coronary events. Both TNF-a and IL-6 wereelevated in the coronary sinus compared with theaortic root in patients with UAP. There is anintracardiac inflammatory response in UAP thatappears to be the result of low-grade myocardialnecrosis. Mizia-stec et al . stated that serum concen-trations of TNF-a were significantly higher in patientswith CAD than in the control group.24

In our study, TNF-a was significantly higher in thestudy group than in the control group. Although wedid not measure troponin, we speculated that thisincrease might be associated with microinfarcts inUAP patients. Thus, we thought that elevated TNF-acould be used for risk stratification.

Limitations of this study

Unfortunately, our study contained a relatively smallnumber of patients to reach an exact conclusion. Thissituation is valid especially for evaluations of IL-8 andIL-2 levels. Another limitation was that we acceptedthe chest pain commencement time within 6 h(approximately 4.89/1.2 h) for the UAP group.Some factors such as taking serum samples atadmission of the patients with UAP and changes ofcytokines according to time passed, not measuringthe troponin level that is an indicator of microinfarctsin patients with UAP and high-risk patients, and notdetermining its relation to other related parametersare the other restrictions.

In conclusion, we suggest that IL-1b, IL-8 and TNF-a are directly involved in the triggering stage of acutecoronary events. As for CRP, high levels of inflam-matory cytokines such as IL-1b and TNF-a may beused for determining the high-risk patients in UAP.

References

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Received 5 September 2003Accepted 22 September 2003

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Mediators of Inflammation � Vol 12 � 2003 365

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