Onno J. de Boer

Leucocyte recruitment in rupture prone regions of lipid-rich plaques: a prominent role for neovascularization?

OBJECTIVE Microvessels in atherosclerotic plaques provide an alternative pathway for the recruitment of leucocytes in the lesions. The present study was designed to investigate the potential role of these microvessels in creating vulnerable sites in atherosclerotic plaques. METHODS Thirty-four atherosclerotic plaques were obtained from 25 patients undergoing carotid endartherectomy (n = 16), femoral endartherectomy (n = 6) and aortic surgery (n = 12). Plaques were histologically classified as either lipid-rich (rupture prone, n = 21) or fibrous (stable, n = 13). Serial cryostat sections were immunohistochemically investigated using monoclonal antibodies against endothelial cells (ULEX-E and F-VIII), vascular endothelial growth factor (VEGF), endothelial adhesion molecules (ICAM-1, VCAM-1, E-Selectin, CD40) and inflammatory cells (macrophages (CD68) and T lymphocytes (CD3). RESULTS The microvessel density in lipid-rich plaques was significantly increased as compared to fibrous plaques. Most of these vessels were located in the shoulder-region of the plaque and at the base of the atheroma. Microvessels in lipid-rich plaques also expressed increased levels of ICAM-1, VCAM-1, E-Selectin and CD40. Moreover, inflammation was most abundantly present in the proximity of microvessels. VEGF was only observed on vessels and mononuclear cells in lipid-rich plaques, suggesting that this factor may play a role in microvessels formation. CONCLUSIONS Neovascularisation and expression of adhesion molecules by microvessels at sites of vulnerable lipid-rich plaques may sustain the influx of inflammatory cells and hence, could contribute to plaque destabilization.

Overrepresentation of IL-17A and IL-22 Producing CD8 T Cells in Lesional Skin Suggests Their Involvement in the Pathogenesis of Psoriasis

Background Although recent studies indicate a crucial role for IL-17A and IL-22 producing T cells in the pathogenesis of psoriasis, limited information is available on their frequency and heterogeneity and their distribution in skin in situ. Methodology/Principal Findings By spectral imaging analysis of double-stained skin sections we demonstrated that IL-17 was mainly expressed by mast cells and neutrophils and IL-22 by macrophages and dendritic cells. Only an occasional IL-17pos, but no IL-22pos T cell could be detected in psoriatic skin, whereas neither of these cytokines was expressed by T cells in normal skin. However, examination of in vitro-activated T cells by flow cytometry revealed that substantial percentages of skin-derived CD4 and CD8 T cells were able to produce IL-17A alone or together with IL-22 (i.e. Th17 and Tc17, respectively) or to produce IL-22 in absence of IL-17A and IFN-γ (i.e. Th22 and Tc22, respectively). Remarkably, a significant proportional rise in Tc17 and Tc22 cells, but not in Th17 and Th22 cells, was found in T cells isolated from psoriatic versus normal skin. Interestingly, we found IL-22 single-producers in many skin-derived IL-17Apos CD4 and CD8 T cell clones, suggesting that in vivo IL-22 single-producers may arise from IL-17Apos T cells as well. Conclusions/Significance The increased presence of Tc17 and Tc22 cells in lesional psoriatic skin suggests that these types of CD8 T cells play a significant role in the pathogenesis of psoriasis. As part of the skin-derived IL-17Apos CD4 and CD8 T clones developed into IL-22 single-producers, this demonstrates plasticity in their cytokine production profile and suggests a developmental relationship between Th17 and Th22 cells and between Tc17 and Tc22 cells.

Low numbers of FOXP3 positive regulatory T cells are present in all developmental stages of human atherosclerotic lesions.

Background T cell mediated inflammation contributes to atherogenesis and the onset of acute cardiovascular disease. Effector T cell functions are under a tight control of a specialized T cell subset, regulatory T cells (Treg). At present, nothing is known about the in situ presence of Treg in human atherosclerotic tissue. In the present study we investigated the frequency of naturally occurring Treg cells in all developmental stages of human atherosclerotic lesions including complicated thrombosed plaques. Methodology Normal arteries, early lesions (American Heart Association classification types I, II, and III), fibrosclerotic plaques (types Vb and Vc) and ‘high risk’ plaques (types IV, Va and VI) were obtained at surgery and autopsy. Serial sections were immunostained for markers specific for regulatory T cells (FOXP3 and GITR) and the frequency of these cells was expressed as a percentage of the total numbers of CD3+ T cells. Results were compared with Treg counts in biopsies of normal and inflammatory skin lesions (psoriasis, spongiotic dermatitis and lichen planus). Principle findings In normal vessel fragments T cells were virtually absent. Treg were present in the intima during all stages of plaque development (0.5–5%). Also in the adventitia of atherosclerotic vessels Treg were encountered, in similar low amounts. High risk lesions contained significantly increased numbers of Treg compared to early lesions (mean: 3.9 and 1.2%, respectively). The frequency of FOXP3+ cells in high risk lesions was also higher compared to stable lesions (1.7%), but this difference was not significant. The mean numbers of intimal FOXP3 positive cells in atherosclerotic lesions (2.4%) was much lower than those in normal (24.3%) or inflammatory skin lesions (28%). Conclusion Low frequencies of Treg in all developmental stages of human plaque formation could explain the smoldering chronic inflammatory process that takes place throughout the longstanding course of atherosclerosis.

Differential expression of interleukin-17 family cytokines in intact and complicated human atherosclerotic plaques†

In addition to the classical TH1 and TH2 cytokines, members of the recently identified IL‐17 cytokine family play an important role in regulating cellular and humoral immune responses. At present nothing is known about the role of these cytokines in atherosclerosis. Expression of IL‐17A, ‐E and ‐F was investigated in atherosclerotic tissue by rtPCR and immunohistochemistry. IL‐17E and its receptor were further studied in cultured smooth muscle cells and endothelial cells, using rtPCR and western blot. rtPCR showed that IL‐17A, ‐E and ‐F were expressed in the majority of plaques under investigation. IL‐17A/F was expressed by mast cells in all stages of plaque development. IL‐17A/F+ neutrophils were always observed in complicated plaques, but hardly in intact lesions. IL‐17A/F+ Tcells (‘TH17’) were never observed. IL‐17E was expressed by smooth muscle cells and endothelial cells in both normal and atherosclerotic arteries, and in advanced plaques also extensively by mature B cells. Cultured smooth muscle cells and endothelial cells were found to express both IL‐17E and its functional receptor (IL‐17RB). The constitutive expression of IL‐17E by resident plaque cells, and the additional presence of IL‐17E+ B cells and IL‐17A/F+ neutrophils in advanced and complicated plaques indicates a complex contribution of IL‐17 family cytokines in human atherosclerosis, depending on the stage and activity of the disease. Copyright

Adventitial infiltrates associated with advanced atherosclerotic plaques: structural organization suggests generation of local humoral immune responses.

Advanced atherosclerotic lesions often contain adventitial lymphoid infiltrates, which occasionally contain nodular aggregates resembling lymphoid follicles. The structural organization suggests that local maturation of B cells may take place at these sites, as described for the mucosa‐associated lymphoid tissue (MALT). This concept was evaluated by studying the micro‐anatomy and cellular composition of adventitial infiltrates associated with advanced atherosclerosis of the aorta. Sections of 22 atherosclerotic aortas were stained immunohistochemically for cellular markers characteristic for lymphoid follicles, such as HECA‐452‐positive endothelial cells, CD20‐positive B cells, CD21‐positive follicular dendritic cells, and CD68‐positive macrophages. Ki‐67 was used as a proliferation marker. The TUNEL technique was used to study the presence of apoptotic cells. Specimens containing MALT served as comparison and positive controls. Seven of the 22 atherosclerotic aortas contained adventitial infiltrates resembling lymphoid follicles. The organized nodular centres were composed of CD45RA+ B cells, follicular dendritic cells (CD21+), a few T lymphocytes (CD3+) and ‘tingible body’ macrophages (CD68+). A large number of cells were Ki‐67‐positive; apoptotic bodies were numerous and phagocytosed by macrophages. The parafollicular area contained CD45RO‐positive T cells and HECA‐452‐positive vessels. Vessels elsewhere were always HECA‐452‐negative. Specimens with MALT showed similar features. This study reveals a close resemblance between adventitial lymphoid infiltrates in advanced atherosclerotic aortic disease and MALT, suggesting local generation of a humoral immune response, likely to be initiated by antigens released during a process of long‐standing tissue injury and inflammation as part of advanced atherosclerosis. Copyright

Atherosclerosis, inflammation, and infection

In recent years, it has been shown that inflammation plays an important role in the pathogenesis of atherosclerosis. Activated macrophages, T lymphocytes, and mast cells are present in atherosclerotic plaques, which has led to the notion that the inflammatory response is an immune‐mediated process. Complicated lesions, moreover, appear to be associated with an increase in the amount of the inflammatory response and in these patients, increased levels of acute phase proteins are present. The appreciation that atherosclerosis is an immune‐mediated inflammatory disease has also led to renewed interest in the potential role of infectious agents in initiating or modulating atherosclerosis. Seroepidemiological studies have shown raised antibody titres against several micro‐organisms. However, as yet, there are hardly any data available that provide a sound scientific basis for an infectious origin. Of all potential candidate organisms, Chlamydia pneumoniae appears as the one most likely involved in atherogenesis. C. pneumoniae has been retrieved from atherosclerotic tissues; the level of raised plasma titres correlates with the severity of symptomatic atherosclerotic disease; and the incidence of C. pneumoniae‐responsive T cells in peripheral blood is increased in patients with coronary heart disease. It also appears that in some patients T cells generated from atherosclerotic plaques respond to C. pneumoniae. At the present state of knowledge, however, it is fair to state that the relationship between infection, intraplaque inflammation, and atherosclerosis still remains hypothetical, despite the increasing evidence that such a relationship could exist. Copyright

Increased expression of T cell activation markers (CD25, CD26, CD40L and CD69) in atherectomy specimens of patients with unstable angina and acute myocardial infarction

Atherosclerotic plaques contain a chronic immune mediated inflammation in which T cells play an important role. A previous study revealed that the numbers of interleukin-2 receptor-positive T cells is increased in culprit lesions of patients with acute coronary syndromes; a finding of considerable interest since it indicates a recent change in the intraplaque T cell mediated immune response. Confirmation of this observation is important, because it could provide insight into the onset of the acute event. We have, therefore, expanded our earlier work by using a panel of different T cell activation markers (CD25, CD26, CD40L, CD69). The study is based on 58 culprit lesions from patients who underwent coronary atherectomy. There were four groups of patients: chronic stable angina (n=13), stabilized unstable angina (n=16), refractory unstable angina (n=15), and acute myocardial infarction (AMI; n=14). Activated T cells were expressed as a percentage of the total of CD3-positive cells. CD25, CD26, CD40L, and CD69/CD3 percentages increased with the severity of the coronary syndrome. In patients with AMI all percentages were significantly higher than in patients with chronic stable angina. CD25, CD26, CD40L, and CD69/CD3 percentages in patients with an unstable condition (refractory unstable angina and AMI) were significantly higher than those in patients with a stable condition (chronic stable or stabilized unstable angina) The finding that the percentage of T cells with recent onset activation is significantly increased in the culprit lesions of patients with acute coronary syndromes suggests strongly that a recent change in pathogenic stimulation has occurred leading to local T cell activation.

Immunohistochemical Analysis of Regulatory T Cell Markers FOXP3 and GITR on CD4+CD25+ T Cells in Normal Skin and Inflammatory Dermatoses

Regulatory T cells (Treg) are a subset of T lymphocytes that play a central role in immunologic tolerance and in the termination of immune responses. The identification of these cells in normal and inflammatory conditions may contribute to a better understanding of underlying pathology. We investigated the expression of FOXP3 and GITR in normal skin and in a panel of different inflammatory dermatoses. Immunohistochemical double stainings in skin tissue sections revealed that FOXP3 and GITR were almost exclusively present on T cells that express both CD4 and CD25. Further, immunohistochemical double staining, as well as fluorescence-activated cell sorter analysis, on peripheral blood T cells showed that most FOXP3+ cells expressed GITR and vice versa, whereas a minority were single-positive for these markers. The mean frequency of FOXP3+ T cells in spongiotic dermatitis, psoriasis, and lichen planus was in the same range (25-29%), but the frequency of these cells in leishmaniasis appeared to be lower (∼15%), although this was not statistically significant. The mean frequency of GITR+ T cells was fairly similar in all conditions studied (14-20%). Normal human skin also contained FOXP3+ and GITR+ cells in the same frequency range as in diseased skin, but the absolute numbers were, of course, much lower. In conclusion, frequencies of FOXP3+ and GITR+ T cells were similar in all inflammatory skin diseases studied and normal skin, despite the well-known differences among the inflammatory conditions under investigation.

Cytokine secretion profiles of cloned T cells from human aortic atherosclerotic plaques

T cells take part in the chronic inflammatory reaction in atherosclerotic plaques, but their specific role in atherosclerosis has not yet been fully elucidated. Nevertheless, one may anticipate that activated T cells may secrete cytokines capable of modulating the morphology and hence the stability of plaques by regulating cell proliferation, lipid metabolism, and extracellular matrix (ECM) synthesis and/or degradation. This study has been designed to investigate the functional properties of T cells in atherosclerotic lesions. For this purpose, T‐cell clones were generated from atherosclerotic plaques isolated from human aortas obtained at autopsy from six subjects. Cloned cells were activated with PMA and OKT‐3 to initiate cytokine production and cytokine profiles of CD4‐positive clones were measured by ELISA. The majority of the T‐cell clones (125/155, 81 per cent) produced both interferon (IFN)‐γ and interleukin (IL)‐4 (type 0 cytokine profile). Moreover, the production of IFN‐γ was dominant in the majority of these clones. A type 1 cytokine profile (high levels of IFN‐γ and low levels of IL‐4) was found in 17 per cent of the clones (27/155). Only three clones (2 per cent) showed a type 2 cytokine secretion pattern (high levels of IL‐4 and low levels of IFN‐γ). No cytolytic activity could be established in plaque‐derived T cells. Our results show that the T‐cell population in atherosclerotic lesions is heterogeneous, but the most dominant T cell by far is the one with a type 0 cytokine profile. The dominant secretion of IFN‐γ by T‐cell clones suggest an important role for plaque T cells in modulating the growth and differentiation of other cells, such as macrophages and smooth muscle cells in atherosclerotic plaques. Copyright

Costimulatory molecules in human atherosclerotic plaques: an indication of antigen specific T lymphocyte activation

Atherosclerotic plaques contain inflammation, composed largely of macrophages and lymphocytes. A proportion of lymphocytes shows signs of activation, but the question arises whether they are activated in an antigen specific way. The expression of costimulatory molecules-receptors that provide accessory signals during antigen-specific activation is a prerequisite for such a condition. This aspect of inflammation in atherosclerotic lesions has not been investigated. Human arterial segments with diffuse intimal thickening, fatty streaks and atherosclerotic plaques were studied with immuno-single and double staining methods. Macrophages and T lymphocytes were stained with CD68 and CD3, respectively, and pan-B cell markers CD19 and CD22 were also used. Costimulatory molecules B7-1 and B7-2, together with their common ligand CD28, and CD27 with its ligand CD70, were stained with specific monoclonal antibodies. The results show that most T lymphocytes were CD27 positive and that only a subpopulation of these (5-15%) was positive also for B7-1, CD28 and CD70. Macrophages expressed B7-1, B7-2, CD28 and CD70, while macrophages positive for CD28 and CD70 have not been reported yet. The expression of costimulatory molecules was most pronounced in the superficial layers at the fibrous cap, but decreased towards the lipid core. This study shows, therefore, that atherosclerotic plaques provide costimulatory signals generally accepted as a prerequisite for adequate T cell stimulation. In addition, this study reveals that only approximately 5-15% of the lymphocytes appears actively involved in the inflammatory reaction.