Results

Histopathological analysis showed that all specimens contained fibrous tissue, either fibrocellular or fibrosclerotic, or both. The number of lesions containing thrombus and atheroma was highest in patients with refractory unstable angina or acute myocardial infarction. There were no differences in total tissue areas of sections among the four patient groups (table 1).

Immunohistochemical examination showed that all lesions contained CD3 positive T cells, which were frequently seen in clusters in fibrocellular tissue and the border zones of atheromas (fig 1). Variable numbers of scattered T cells were also present throughout the tissues. Thrombus and extracellular lipid fragments (atheroma) contained few, if any, lymphocytes. Medial fragments were devoid of T cells. The mean (SEM) numbers of CD3 positive T cells per mm² increased from patient group 1 (stable angina: 14.7 (1.8)) to groups 2 (Braunwald class I, II: 23.1 (2.6)) and 3 (Braunwald III: 45.1 (8.8)). In patient group 4 (acute myocardial infarction), however, the mean (SEM) number of CD3 positive cells was lower (16.5 (2.3)) (p_1–3, p_2–3, and p_3–4< 0.05).

• Download figure
• Open in new tab
• Download powerpoint

Figure 1

(A) Haematoxylin and eosin stained section of a specimen from a patient with Braunwald type III unstable angina showing fibrocellular tissue (f), thrombus (t), atheroma (a), and a fresh clot related to atherectomy (c). (B) Anti-CD3 stained section adjacent to panel A shows a cluster of T cells and scattered immunopositive cells throughout the fibrocellular tissue.(C) Higher power magnification of the boxed area of (A) stained with anti-CD3, showing the ring shaped staining pattern of all T cells (CD3 positive). (D) Higher power magnification of the boxed area of (A) showing a subpopulation of IL-2R positive cells stained with anti-CD25. The yellow granules are ceroid pigment, an end product of lipid oxidation.

CD25 positive T cells were also found in clusters, although in much fewer numbers. Moreover, they were present in only a few lesions. Fifty two per cent of the total number of lesions of patients with chronic stable angina contained CD25 positive T cells. Patients with unstable angina (group 2: 77.8% of lesions and group 3: 90.9%) and myocardial infarction (89.4%) had a much higher incidence of lesions with CD25 positive T cells. The relative number of activated T lymphocytes (CD25/CD3 ratios calculated for each specimen) increased from patient groups 1 (0.022 (0.009)) and 2 (0.033 (0.007)) to groups 3 (0.078 (0.003)) and 4 (0.185 (0.054)) (p_1–4 and p_2–4< 0.05) (fig 2).

• Download figure
• Open in new tab
• Download powerpoint

Figure 2

Mean (SEM) numbers of T cells (CD3 positive) per mm² atherectomy tissue and percentages of activated (CD25 positive) T cells (CD25/CD3 ×100) for each patient group. Group 1, stable angina; group 2, stabilised unstable angina; group 3, refractory unstable angina; group 4, acute myocardial infarction.*p_1–3, p_2–3, and p_3–4< 0.05. **p_1–4 and p_2–4 < 0.05 (post hoc test).

Discussion

Increased numbers of inflammatory cells, including macrophages, T lymphocytes, and mast cells, in atherosclerotic plaques are associated with severity of the acute coronary syndrome.9 ,10 The present study also shows an increase in recent activation of the T cell response. Other studies have reported the presence of several activation markers on T cells in human atherosclerotic plaques. A large proportion of T cells within randomly sampled plaques present the very late activation antigen VLA-1 and HLA-DR molecules, whereas relatively low numbers express IL-2R.5 This suggests long term low level activation in atherosclerosis.15 These studies, however, were carried out on randomly selected, mostly uncomplicated plaques, and none relates these findings to clinical status.3-5 In this study the percentage of IL-2R positive (CD25 positive) T cells increased in atherectomy specimens obtained from patients with refractory unstable angina (7.8% of the total number of (CD3 positive) T cells) or acute myocardial infarction (18.5%) compared with the relatively low percentages in specimens from patients with stable angina (2.2%) or stabilised unstable angina (3.3%).

Expression of IL-2R is an important marker for T cell activation, as it indicates recent stimulation of these cells in a cell mediated immune response. IL-2R appears on the surface of T cells shortly (within 2–24 hours) after cytokine stimulation by macrophages and persists for only a few days after diminution of the stimulating agents.16During this period a variety of inflammatory mediators are produced, particularly by cytokine activated macrophages and T cells themselves. A causal role for the local burst of inflammatory activity and the onset of acute syndromes seems likely, as inflammatory products secreted during amplification of the immune response may have detrimental effects on stability of the fibrous skeleton and integrity of the plaque endothelium and are also capable of increasing local vasomotor activity of the vessel wall.17 ,18 It seems therefore that T cell activation is another feature of the complex of biological processes associated with plaque destabilisation.

Raised levels of systemic markers for inflammation, such as C reactive protein,19 ,20 and activated circulating monocytes21 have been reported in patients with severe unstable angina. Recently, Serneri and colleagues22 found increased numbers of HLA-DR positive T cells and increased levels of soluble IL-2R (S IL-2R) in the serum of patients with unstable angina. These variables gradually decreased over a period of eight weeks after the coronary event, indicating that acute T cell activation can be detected in serum around the time of onset of coronary syndromes. Systemic markers for lymphocyte activation, however, may not necessarily relate to the situation in the atherosclerotic plaque, and may arise from another inflammatory (infectious) disease.23

The relatively low number of total (CD3 positive) T cells in specimens from patients with acute myocardial infarction was surprising. This finding probably relates to the substantially larger amounts of thrombus, practically devoid of T cells, that were found. The ratio of activated T cells versus the total number of T cells, the prime interest of this study, however, is not affected by thrombus.

Immunocytochemical detection of IL-2R on T lymphocytes is considered to be a strong indication that recent antigenic stimulation has occurred. Indeed, the microenvironment of atherosclerotic plaques contains additional components necessary for antigenic stimulation—for example, HLA-DR positive macrophages, which potentially may serve as antigen presenting cells,2 and sets of co-stimulatory molecules and their ligands on macrophages and T cells.24Moreover, in situ studies of human plaque tissue have shown that cytokines produced by inflammatory cells include γ interferon and interleukin 12, but virtually no interleukin 4.25 ,26These findings indicate a preference for a cell mediated immune response in atherosclerotic plaques.

Recognition of increased recent activation of the immune response in plaques underlying acute coronary events emphasises further the need to identify those antigens responsible for triggering the process. Of several potential antigens that have been suggested, including heat shock proteins,27 ,28 neoantigens on cytomegaly virus infected cells,29 chlamydial antigens,30 and lipid derivatives,6 ,31 ,32 antigenic determinants of oxidised low density lipoproteins (oxLDL) are of particular interest. So far, oxLDL is the only antigenic structure capable of stimulating human plaque derived T lymphocytes in vitro.6 Moreover, cellular analysis of atherosclerotic lesions in apolipoprotein E knockout mice, performed by the same researchers, has shown participation of CD25 positive, CD4 positive cells in lesion formation in these genetically hypercholesterolaemic animals. The authors speculated that there might be a direct link between accumulation of cholesterol in the vessel wall and activation of T cells, possibly by autoimmune responses to modified lipoproteins.32Interestingly, we regularly observed clusters of lymphocytes in close proximity to ceroid pigments, which are considered to be end products of lipid oxidation in plaque tissue (fig 1). If derivatives of oxLDL are responsible for activation of recent immune responses in plaques, either solely or in combination with other antigens, then patients with symptomatic coronary artery disease should be managed from a different perspective. Lipid lowering strategies may be beneficial not only because they can decrease the lipid mass of plaques as a long term effect, but also because they can reduce critical levels to induce a “burst” of recent inflammation and, hence, the incidence of acute coronary events.19 ,20