Basic Research in Cardiology

, Volume 104, Issue 3, pp 341–351

Beta2-integrin activation on T cell subsets is an independent prognostic factor in unstable angina pectoris

Authors

    • Dept. of CardiologyRuprecht-Karls-University
  • Hülya Aksoy
    • Dept. of CardiologyRuprecht-Karls-University
  • Guido H. Wabnitz
    • Institute for ImmunologyRuprecht-Karls-University
  • Christian Volz
    • Dept. of CardiologyRuprecht-Karls-University
  • Christian Erbel
    • Dept. of CardiologyRuprecht-Karls-University
  • Henning Kirchgessner
    • Institute for ImmunologyRuprecht-Karls-University
  • Evangelos Giannitsis
    • Dept. of CardiologyRuprecht-Karls-University
  • Hugo A. Katus
    • Dept. of CardiologyRuprecht-Karls-University
  • Yvonne Samstag
    • Institute for ImmunologyRuprecht-Karls-University
  • Thomas J. Dengler
    • Dept. of CardiologyRuprecht-Karls-University
ORIGINAL CONTRIBUTION

DOI: 10.1007/s00395-008-0770-8

Cite this article as:
Konstandin, M.H., Aksoy, H., Wabnitz, G.H. et al. Basic Res Cardiol (2009) 104: 341. doi:10.1007/s00395-008-0770-8

Abstract

Background

Cardiac troponins provide excellent risk stratification in unstable angina (UA), but no reliable markers are available in troponin-negative patients. Beta2-integrin mediated T cell recruitment plays a pivotal role in coronary atherosclerotic plaque rupture. The present study investigates beta2-integrin activation on T cell subsets as a risk marker in UA.

Methods

Functional activation (affinity/avidity) of beta2-integrins on T cells was measured using a flow cytometry-based whole blood assay in 87 patients with UA.

Results

Beta2-integrin activation was significantly higher in patients with severe coronary artery disease (sC) and myocardial infarction (MI) compared to patients with no/minimal coronary atherosclerosis (no/mC), irrespective of troponin status. Adjusted for cardiovascular risk factors, medication, left ventricular function, MI at enrollment and high sensitivity C-reactive protein (hsCRP), beta2-integrin activation was independently associated with incidence of revascularization, hospitalization and all major cardiovascular events during 9 months of follow-up after index investigation. The highest prognostic value of beta2-integrin activation was seen in troponin-and hsCRP-negative patients.

Conclusion

Quantitative assessment of T cell beta2-integrin activation allows to identify high risk patients with UA and sC without established MI; furthermore, it is associated with incidence of future cardiovascular events independent of conventional risk factors (troponin, hsCRP).

Keywords

acute coronary syndromeT cellinflammationintegrinatherosclerosis

Introduction

Coronary artery disease (CAD) is considered as chronic inflammatory process within the arterial wall resulting in narrowing of the vessel lumen. In earliest detectable lesions accumulation of leukocytes can be observed. Once an atherosclerotic plaque has developed, it either presents as stable plaque characterized by thick fibrous cap with few inflammatory infiltrates and small lipid core or as an unstable plaque with thin fibrous cap, large lipid core and many inflammatory cells. The latter has the potential to rupture, bringing subendothelial structures in contact with components of the coagulation cascade and initiating thrombus formation [10, 12, 25, 45, 47]. Clinically, this process occurs as acute coronary syndrome (ACS) with symptoms of unstable angina pectoris (UA). Such patients, however, represent a heterogeneous cohort regarding findings in coronary angiography: The spectrum reaches from no detectable atherosclerotic changes within coronary tree to severe 3-vessel disease with myocardial infarction (MI) due to occlusion of a coronary artery branch [2]. Cardiac Troponins (cTnT, cTnI) are highly specific and sensitive markers for myocardial cell damage allowing discrimination of MI from UA without ongoing myocardial necrosis [11, 19, 31, 41].

T-helper cells play a pivotal role in the acute exacerbation of the chronic, smoldering inflammatory process within the atherosclerotic plaque. Recruited IFN-γ-secreting T cells activate macrophages within the plaque to release matrix metallo-proteases and other lytic enzymes. Thereby, the extracellular matrix of the fibrous cap becomes degraded, destabilized and prone to rupture [13, 29]. Post-mortem analyses have shown increased numbers of activated T cells within the culprit plaque of patients with ACS. Changes in the composition of the T-helper cell compartment in peripheral blood have been described in patients with UA. Especially, IFN-γ-secreting T-helper cells lacking the costimulatory molecule CD28 (CD4+ CD28null cells) were found in peripheral blood of patients with UA [28, 34, 37, 43].

T-helper cells can be differentiated functionally and phenotypically into six subsets according to the expression of CD28, CD45RA and CCR-7 [3]. To exercise their effect on plaque destabilization, T-helper cells need to be recruited to the plaque following a multi-step-process of transendothelial migration, which is critically dependent on the activation and adhesive properties of integrins on leukocytes, interacting with their respective ligands on endothelium (ICAM-1) [38].

The pivotal increase in leukocyte adhesiveness is achieved through an increase in affinity and/or avidity of the integrins [18, 40]. Recently, we established a flow cytometry-based assay allowing quantification of integrin activation in leukocytes in whole blood. This test relies on preformed soluble ICAM-1 complexes and is thus called ligand-complex-based adhesion assay (LC-AA). With this approach both mechanisms of adhesiveness, affinity and avidity, can be detected and integrin activation can be quantified on single cell level in a small sample of whole blood [22, 23].

T-helper cell recruitment into the plaque precedes plaque destabilization and thus MI. Since activation of beta2-integrins is essential for this process, aim of the present study was to test the diagnostic and prognostic value of beta2-integrin activation in various T cell subsets as novel functional biomarker in patients with UA.

Methods

Patients

Between March and August 2006, patients presenting with UA at University Hospital Heidelberg were screened for participation in this study. Other inclusion criteria were age > 18 and <80 years and written informed consent under a protocol approved by the institutional review board in accordance with the declaration of Helsinki. One hundred and thirty-one patients met the inclusion criteria. Exclusion criteria were immunosuppressive medication(14), acute infection(5), inflammatory co-morbidity(3), other organic heart disease (cardiomyopathy or valvular diseases(6)) and lack of coronary angiography during hospital stay(16). Eighty-seven patients were included and divided into 4 groups: (1) No or minimal CAD (no/mC): no elevation of cardiac troponin T (cTnT) and no coronary artery lesion exceeding stenosis of 25%. (2) Intermediate CAD (iC): cTnT-negative patients with coronary artery stenosis of >25% and <75% in one or several vessels. (3) Patients with severe CAD (sC) that were cTnT negative and had at least one stenosis of ≥75%. (4) MI: cTnT positive and exclusion of other non-ischemic reasons for elevated cTnT (myocarditis, pulmonary embolism, reviewed in [24]). In the MI group one patient had a maximal coronary artery stenosis of 50–75%, all other patients of this group showed at least one coronary artery stenosis of ≥90%. Serum concentration of cTnT ≥0.03 µg/l was considered as elevated value, i.e. positive test result. Study group assignment occurred blinded to flow cytometry results of integrin activation. cTnT was measured by quantitative immunoassay based on chemiluminescence (Elecsys TroponinT, Roche Diagnostics). High sensitivity C-reactive protein (hsCRP) was analysed with nephelometry (Nephelometer-analyzer, Dade Behring (Schwalbach)), using diagnostic cut-off level of 10 mg/l [15, 33].

Coronary angiography

Examination was performed according to guidelines for coronary angiography of the German society for Cardiology [36]. Angiograms were evaluated off-line. Investigators were uninformed/blinded to results of this study. Left ventricular function estimated by angiography was coded semi-quantitatively: good LV function: 4 points, mildly reduced function: 3 points, intermediate reduction of LV function: 2 points and severe reduction of LV function: 1 point.

Flow cytometric analysis of T cell adhesiveness

Analysis of spontanous beta2-integrin activation of T cell subsets was performed using the LC-AA[23]. In brief, 40 µl whole blood was transferred to 37°C. Per sample 2 µl anti-CCR7-Pe-Cy7 was added into the whole blood. This antibody had to be added before fixation since the epitope was not detected properly, when the staining has been performed after fixation. Addition of antibody before fixation has been shown to have no influence on T cell function (data not shown). Subsequently, 8 µl of preformed Fitc-labeled ICAM-1-Fc-F(ab)2 complexes and 14 µl PBS containing 5 mM Mg2+ were added. After 30 min 10 volumes of pre-warmed (37°C) FACS-Lysing solution (BD; Pharmingen) supplemented with 2 mM Mg2+/1 mM Ca2+ was added, samples were vortexed and incubated for 5 min at 37°C. Fixation was stopped by adding ice-cold FACS buffer (PBS supplemented with 0.05% sodium azid, 0.5% BSA and 5% FCS). After centrifugation cells were stained for subpopulations (CD3-APC-Cy7, CD4-PerCp, CD28-PE, CD45RA-APC) and analyzed by flow cytometry (FACS-Canto, BD). All antibodies used were from BD, Pharmingen. To generate ICAM-1-Fc-F(ab)2 complexes, FITC-labeled goat anti-human IgG Fcγ specific F(ab)2 fragments (in a 1/6.25 dilution, Jackson ImmunoResearch) and ICAM-1-Fc (200 µg/ml) were incubated overnight at 4°C in PBS. Binding of FITC-labeled complexes to cells has been analysed by quantification of mean fluorescence intensity (MFI, dimensionless value) per single cell. Specificity of complex binding to beta2-integrins on T cells has been shown recently [23]. PBS was from Cell Concepts, all other reagents used were from Sigma.

Patient blood samples were taken immediately after admission and strictly before coronary angiography to avoid stimulation of cells by treatment of the patient (drugs, contrast agent, etc.). Samples were stored at room temperature; experiments were performed within 4 h after venous puncture. These conditions were tested to have no influence on function of beta2-integrin-mediated adhesiveness of T cell subsets.

Follow-up

Endpoint follow-up was performed by interviewing patients directly or their respective general practitioner/cardiologist. Additionally, patients’ files of subsequent hospitalization were analyzed. Mean follow-up time was 9.06 months. Pre-specified endpoints were (1) death of any cause, (2) death due to a cardiovascular event, (3) recurrence of ACS, (4) revascularization procedure, (5) hospitalization due to cardiovascular events and (6) combined endpoint consisting of any of the major cardiovascular events from (2) to (5) (MACE).

Statistics

For comparisons between two groups the non-parametric Mann–Whitney-test and between ≥3 groups the Kruskall-Wallis with Dunn’s post-test analysis were applied using Graph Pad Prism 4.0, version 2003 software. To test association of T-helper cell adhesiveness with cardiovascular events during follow-up proportional hazard cox regression analysis was performed using SPSS 13.0. For Kaplan-Meier curves differences were analysed by log rank test (SPSS 13.0). P-value < 0.05 was considered statistically significant.

Results

A total of 87 patients were included in the present study. According to coronary angiographic findings and cTnT levels patients were grouped into no/mC, iC, sC and MI. Table 1 shows clinical characteristics of all patients. Patients with MI showed reduced left ventricular function by semi-quantitative analysis (MI 2.6 ± 0.1 Vs. no/mC 3.72 ± 0.1; P < 0.001), elevated leukocyte count (MI 10.9 ± 0.5 cells/µl Vs. no/mC 8.0 ± 0.6 cells/nl; P < 0.01) and increased proportion of statin medication compared to no/mC (MI n = 42 of 44 (93.3%) Vs. no/mC n = 9 of 16 (56.3%); P < 0.01). The incidence of hyperlipoproteinemia was higher in sC patients (n = 13 of 16 (81.3%)) compared to no/mC (n = 7 of 16 (43.8%); P < 0.05). All other parameters (age, gender, other CAD medication, other risk factors) were not different between groups.
Table 1

Clinical characteristics

 

Total (n = 87)

no/m C (n = 16)

iC (n = 10)

sC (n = 16)

MI (n = 45)

Age (years)

64.7 ± 1.1

60.3 ± 2.2

63.6 ± 3.4

66.1 ± 2.3

65.9 ± 1.6 NS

Gender n (%)

  Female

31 (35.6)

8 (50)

5 (50)

3 (18.8)

15 (33.3) NS

  Male

56 (64.4)

8 (50)

5 (50)

13 (81.2)

30 (66.7)

Cardiovascular risk factors n (%)

  Arterial hypertension

74 (85.1)

13 (81.3)

8 (80)

14 (87.5)

39 (86.7) NS

  Hyperlipidemia

48 (55.2)

7 (43.8)

8 (80)

13 (81.3)

20 (44.4)*

  Obesity

32 (36.8)

3 (18.8)

5 (50)

4 (25)

20 (44.4) NS

  Diabetes

20 (23)

4 (25)

0 (0)

5 (31.3)

11 (24.4) NS

  Familial history of CAD

34 (39.1)

8 (50)

4 (40)

7 (43.8)

15 (33.3) NS

  Smoking

43 (49.4)

7 (43.8)

5 (50)

9 (56.3)

22 (48.9) NS

Medication n (%) statins

73 (83.9)

9 (56.3)

8 (80)

14 (87.5)

42 (93.3)**

  Aspirin

83 (95.4)

15 (93.8)

9 (90)

15 (93.8)

44 (97.8) NS

  Beta-blocker

78 (89.7)

13 (81.3)

7 (70)

15 (93.8)

43 (95.6) NS

  ACE-inhibitor or angiotensin-receptor blocker

79 (90.8)

12 (75)

10 (100)

16 (100)

41 (91.1) NS

LV functiona

3.03 ± 0.1

3.72 ± 0.1

3.65 ± 0.2

3.25 ± 0.2

2.6 ± 0.1***

Leukocyte count (cells/nl)

9.5 ± 0.3

8.0 ± 0.6

7.4 ± 0.8

8.3 ± 0.5

10.9 ± 0.5**

Patients with MI showed elevated leukocyte count, increased prescription of statins, reduced LV function compared to patients with no/mC

*P < 0.05; **P < 0.01; ***P < 0.001

aLV function estimated by angiography: good LV function 4 points, mild reduced function 3 points, intermediate reduction of LV function 2 points and severe reduction of LV function 1 point

Beta2-integrins are activated on T cell subsets in sC and MI

Figure 1a shows one representative dot-plot displaying distribution of T-helper cell subsets according to their expression of CD45RA and CCR-7, which allows differentiation of four compartments. Starting in the upper right quadrant these compartments are numbered clock-wise. Naïve T cells express both receptors (I; CD45RA+ CCR-7+), central memory cells lose CD45RA expression but are still CCR-7+ (II; CD45RA- CCR-7+). Effector memory cells are CCR-7 and CD45RA- (III). CD45RA+ CCR-7 cells constitute compartment IV. Cells within compartments III and IV can be differentiated further due to their expression of CD28 in CD28+ (a) and CD28null (b) [3].
https://static-content.springer.com/image/art%3A10.1007%2Fs00395-008-0770-8/MediaObjects/395_2008_770_Fig1_HTML.gif
Fig. 1

Beta2-integrins are activated in patients with sC independent of MI. a 4 subsets of T-helper cells can be differentiated. b compartments III and IV can be sub-divided based on CD28 expression. c Representative blots of scICAM-1 binding on effector memory T cells (IIIa) for no/mC (left) and MI (right). D: individual beta2-integrin-activation of T cells (IIIa) of 87 patients. *P < 0.05

For all six possible subsets spontaneous beta2-integrin activation was quantified using the LC-AA [22, 23]. For all T cell subsets integrin activation was elevated in sC and MI compared to no/mC, most robustly in effector memory cells (IIIa) (Table 2). Representative FACS blots of spontaneous scICAM-1 binding are shown in Fig. 1c for MI and no/mC. The individual distribution of LC-AA integrin activation is shown for effector memory cells (IIIa) in Fig. 1d: in the sC-group MFI increases to 50.6 ± 5.3 (P < 0.05) and in the MI-group to 46.6 ± 2.3 (P < 0.05) compared to a MFI of 34.4 ± 1.3 in no/mC patients. There was no difference between patients with stable CAD and patients without CAD (electronic supplementary material). Table 2 shows the mean ± SD of all subsets. Expression per se of beta2-integrins (CD18) on T cell subsets was not elevated in patients with MI or sC compared to no/mC (not shown).
Table 2

Beta2-integrin activation of T cell subsets precedes MI

T cell subsets (see Fig. 1)

no/m C

iC

sC

MI

I

38.4 ± 2.3

43.9 ± 13.0 NS

52.1 ± 6.7 NS

49.2 ± 2.6 NS

II

38.4 ± 1.3

41.5 ± 6.9 NS

57.6 ± 9.8*

51.4 ± 2.9*

IIIa

34.4 ± 1.3

37.1 ± 7.5 NS

50.6 ± 5.3*

46.6 ± 2.3*

IVa

36.0 ± 2.0

37.1 ± 5.9 NS

43.6 ± 2.8 NS

51.4 ± 3.6**

IIIb

31.8 ± 1.3

27 ± 3.0 NS

38 ± 2.7 NS

39.5 ± 1.8 NS

IVb

33 ± 2.2

82 ± 0 NS

132.9 ± 58.7*

46.1 ± 5.4 NS

The table shows the mean fluorescence intensity (MFI) of spontaneous scICAM-1 binding to T cell subsets. In MI and sC integrins are activated within all subsets independent of myocardial necrosis

*P < 0.05; **P < 0.01

Beta2-integrin activation of effector memory T-helper cells allows prediction of future cardiovascular events

Integrin activation of effector memory cells (IIIa) was selectively analysed based on most robust changes in subpopulation analysis. For 85 out of 87 patients long-term follow-up after index event (including invasive, interventional treatment) was available. Four patients died, all from cardiovascular events; 10 patients suffered repeat ACS during follow-up, 21 patients had to undergo revascularization procedures, 33 patients were hospitalized for cardiovascular events and in 36 patients a combined endpoint consisting of any of the previous events was reached (MACE). Association between integrin activation and future cardiovascular events by univariate cox regression analysis was shown (Table 3), high sensitivity C-reactive protein (hsCRP), a well-accepted prospective risk marker of cardiovascular outcome, was analyzed in parallel yielding less strong statistical correlation. Integrin activation was associated with incidence of future ACS (P = 0.008), revascularization (P = 0.027), hospitalization (P =  0.01) and MACE (P = 0.01). For the established marker hsCRP significant association with hospitalization (P = 0.035) and MACE (P = 0.013) was demonstrated.
Table 3

Univariate analysis of the association of integrin activation of memory T cells (IIIa) and hsCRP with cardiovascular events

Predictor

End point

Hazard ratio (95% CI)

P

Beta2-integrin activation (IIIa)

MACE

1.018 (1.004–1.033)

0.01

Revascularization

1.020 (1.002–1.038)

0.027

Hospitalization

1.019 (1.005–1.034)

0.01

ACS

1.032 (1.008–1.056)

0.008

Death

0.99 (0.928–1.057)

0.763

hsCRP

MACE

2.423 (1.209–4.855)

0.013

Revascularization

1.842 (0.714–4.751)

0.207

Hospitalization

2.227 (1.057–4.690)

0.035

ACS

1.745 (0.451–6.754)

0.420

Death

3.975 (0.560–28.229)

0.168

Cox regression analysis was performed for the occurence of all endpoints MACE, revascularization, hospitalization, occurrence of ACS during follow-up and death

Beta2-integrin activation allows risk prediction in cTnT- and hsCRP-negative patients

For beta2-integrin activation of effector memory cells ROC analysis for MACE was performed (AUC 0.69, CI (0.58–0.81); P = 0.0025). Based on this ROC analysis study population was then dichotomized using a cut-off level of 40.5MFI resulting in 41 patients with high adhesiveness (high LC-AA) and 44 patients with low adhesiveness (low LC-AA). Kaplan-Meier analysis of event-free survival stratified for beta2-integrin adhesiveness was performed (Fig. 2): for MACE (A: P = 0.013), revascularization (B: P =  0.018) and hospitalization (C: P = 0.034) significantly higher incidence of events could be demonstrated with high beta2-integrin activation. For ACS (D) and death (not shown) event curves were not significant different.
https://static-content.springer.com/image/art%3A10.1007%2Fs00395-008-0770-8/MediaObjects/395_2008_770_Fig2_HTML.gif
Fig. 2

Kaplan–Meier curves of event-free survival. Curves display event-free survival and log rank results for MACE (a), revascularization (b), hospitalization (c) and occurrence of ACS (d) depending on beta2-integrin activation

Most notably, the predictive value of beta2-integrin activation was restricted to cTnT- and hsCRP-negative patients (Fig. 3). In the presence of either cTnT or hsCRP elevation beta2-intergrin activation did not serve as additional predictor. In contrast, when at least one of both parameters was negative, integrin activation was able to predict increased risk for all cardiovascular events, results for MACE are shown in Fig. 3. For revascularization (in n = 5 of 41 cTNT-negative patients: log rank 5.032; P = 0.025; in n = 15 of 67 hsCRP-negative patients: log rank 7.575; P = 0.006), hospitalization (in n = 10 of 41 cTNT-negative patients: log rank 12.549; P < 0.001; in n = 23 of 67 hsCRP-negative patients: log rank 6.484; P = 0.011) and occurrence of ACS (in n = 3 of 41 cTNT-negative patients: log rank 6.085; P = 0.014) significant correlation with integrin activation/adhesion became evident (not shown). The strongest effect on risk prediction (MACE) by integrin activation was seen in the subgroup of cTnT- and hsCRP-negative patients (in n = 9 of 36 double negative patients: log rank 13.047; P < 0.001; Fig. 3e).
https://static-content.springer.com/image/art%3A10.1007%2Fs00395-008-0770-8/MediaObjects/395_2008_770_Fig3_HTML.gif
Fig. 3

Predictive value of integrin activation depending on hsCRP and cTnT results. Curves display event-free survival and log rank results for MACE depending on beta2-integrin activation in cTnT-positive (a), cTnT-negative (b), hsCRP-positive (c), hsCRP-negative (d), or cTnT- and hsCRP-double negative patients (e)

To confirm independent risk prediction by beta2-integrin activation, a multivariate cox regression analysis for cardiac events adjusted for both established risk markers hsCRP and cTnT, as well as for age, gender, left ventricular function, classical risk factors (arterial hypertension, hyperlipidemia, obesity, diabetes, familial history of CAD, smoking) and medication (statin, platelet inhibitor, beta-blocker, ACE-inhibitor) was performed. Significant independent association of integrin activation with revascularization (P = 0.015), hospitalization (P = 0.024) and MACE (P = 0.023) could be demonstrated (Table 4). For recurrence of ACS (P = 0.057) a strong trend could be shown, not formally reaching statistical significance.
Table 4

Multivariat cox regression analysis for cardiovascular events

End point (incidence)

Predictor

Hazard ratio (95% CI)

P

MACE (n = 36)

beta2-integrin activation

1.020 (1.003–1.038)

0.023

hsCRP

2.828 (1.241–6.443)

0.013

cTnT

2.363 (1.070–5.218)

0.033

Diabetes

0.305 (0.118–0.791)

0.015

Obesity

2.585 (1.173–5.694)

0.018

LV

0.695 (0.474–1.019)

0.062

PI

0.104 (0.020–0.534)

0.007

Revascularization (n = 21)

Beta2-integrin activation

1.030 (1.006–1.055)

0.015

hsCRP

3.108 (0.994–9.723)

0.051

Gender

0.330 (0.129–0.842)

0.020

PI

0.077 (0.007–0.874)

0.039

Hospitalization (n = 33)

Beta2-integrin activation

1.019 (1.003–1.036)

0.024

hsCRP

2.515 (1.167–5.421)

0.019

ACS (n = 10)

Beta2-integrin activation

1.042 (0.999–1.087)

0.057

cTnT

29.386 (1.42–606.5)

0.029

HLP

78.5 (1.19–5176)

0.041

FA

19.641 (1.54–249.9)

0.022

Death (n = 4)

Beta2-integrin activation

1.140 (0.354–3.670)

0.826

Integrin activation was analysed using mean fluorescence intensity (MFI)—hazard ratio reflects increased ratio for the respective endpoint per increment of the MFI of 1 point. MFI varied between 25 and 119

HLP hyperlipoproteinemia, FA family history for CAD, PI platelet inhibitors, LV left ventricular function, cTnT MI at enrollment

Discussion

Patients with UA represent a heterogeneous cohort regarding findings in coronary angiography and the incidence of future cardiovascular events [2]. Following international guidelines, cardiac troponins are highly sensitive and specific markers for risk stratification in patients with UA defining the gold standard [1, 6]. In the present study we show that beta2-integrin activation of T cells is increased in patients with UA and severe CAD independent of cTnT levels, i.e. apparently preceding myocardial necrosis. In addition, during follow-up it is demonstrated that beta2-integrin activation is associated with the risk of future cardiac events (hospitalization, revascularization, recurrence of ACS and MACE) independent of established risk factors, especially in the subgroup of patients negative for both cTnT and hsCRP.

The main two stimuli resulting in increased T cell adhesiveness are antigen-specific signals via the T cell receptor or cytokine/chemokine signals mediated by G-protein-coupled receptors[9, 21, 30, 44]. For several cytokines increased levels have been shown in patients with UA [7, 20]; also other immune-related antigens are discussed to play a pivotal role in ACS [8, 39, 46]. Beta2-integrin activation of T cells may thus integrate different stimuli resulting into one quantifiable marker, reflecting a generally increased state of inflammation on cellular level. The well-accepted marker for inflammation –hsCRP- also correlates with outcome in patients with UA, but integrin activation was independently associated with the incidence of future events as shown by subgroup analysis of hsCRP- or cTnT-negative patients and multivariate regression analysis including hsCRP and cTnT as covariates. In the present study hazard ratios in the multivariate analysis for established risk factors were comparable with published data, showing that the study collective represents a typical cohort of patients with UA [26]. The hazard ratio for beta2-integrin activation varied between 1.019 for hospitalization and 1.030 for revascularization. Integrin activation was quantified in mean fluorescence intensity (MFI) ranging between 24 and 119 in the present collective. The respective hazard ratio reflects the increased likelihood for the respective endpoint per increment of the MFI of 1 unit. In case of hospitalization therefore the relative risk for occurrence of this endpoint ranges between 1 (MFI 24) to 5.98 (MFI 119). For revascularization the risk varies between 1 (MFI 24) to 16.58 (MFI 119). This range of relative risks indicates the significance of this novel marker compared to established risk factors. Integrin activation seems to represent a more up-stream, early risk indicator in the inflammatory cascade of acute coronary events preceding hsCRP elevation as well as myocardial necrosis (cTnT-positivity). Integrin activation is necessary for extravasation and recruitment of T cells into the plaque resulting in destabilization and vascular inflammation. Rupture of the unstable plaques activates the coagulation cascade leading to thrombo-embolism of the distal coronary circulation and myocardial cell injury, which then leads to elevated troponin levels [5, 13, 16, 17, 29, 35].

So far different soluble serum markers (e.g. cTnT, cTnI, hsCRP, sCD40L), as well as the expression of cellular surface proteins or the level of certain circulating cell subsets (EPC, CD4 CD28null) have been tested for their value as prognostic markers in patients with ACS reflecting the complex pathology of this highly relevant disease [4, 11, 14, 26, 27, 32, 41, 42]. Here we show for the first time, that in addition to expression of proteins the functional activation status of proteins may have prognostic value. Whether affinity, avidity or a combination of both mechanisms is up-regulated in patients with unstable angina cannot be concluded from the present study, since the LC-AA detects both modalities. The advantage of integrin activation as earlier marker preceding myocardial cell damage may have to be counterbalanced with loss of specificity. In other words the herein presented risk model is only valid/applicable after definite exclusion of immunodeficiency, intake of immunosuppressive medication, infection or other inflammatory diseases inducing T cell activation (e.g. arthritis, asthma, inflammatory bowel disease), which may not be trivial in clinical routine. This limitation does, however, apply to numerous other immunologic risk parameters, as well.

In multivariate analysis, beta2-integrin activation of effector memory cells was shown to be a predictive risk factor for hospitalization, revascularization and MACE independent of established factors known to influence cardiovascular outcome; also a strong trend for recurrence of ACS could be shown. The lack of an association regarding ACS and death may be caused by the low incidence and small sample size in the present pilot study. Confirmation in a large collective with prospective analysis is needed.

In summary, beta2-integrin activation on effector memory T cells represents an independent adjunctive prognostic marker in patients with UA—especially in the subgroup of cTnT-negative patients. The data underscore the pathophysiologic relevance of immune cell-mediated processes in cardiovascular disease and extend these concepts to the clinical arena of risk assessment for cardiac events. With the present study an innovative experimental approach derived from a non-traditional inflammation-related pathway—quantifying functional activity on a single cell level—has been identified potentially allowing additional risk stratification in this patient cohort. Specific advantages of beta2-integrin activation appear to lie in earlier/faster detection of risk relevant changes than with acute phase proteins of inflammation (hsCRP) or markers of thrombosis/cardiac injury (cTnT)—thus warranting further analysis of this approach in larger scale studies.

Conflict of interest

None declared.

Supplementary material

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© Steinkopff Verlag Darmstadt 2009