Original article
Circulated CD4⁺CD28^- lymphocytes rate and their cytotoxicity and morphological parameters of internal carotid artery atheromatous plaques in patients with atherosclerosis-related ischemic stroke

Introduction

Inflammatory and immunological factors play an important role in ischemic stroke pathogenesis, taking into consideration their involvement in an ischemic lesion development [9,21].

Over the years, research has also demonstrated the large role of aforesaid factors in the process leading to the acute brain ischemia onset, through development and destabilization of atherosclerotic lesions. It seems that CD4+CD28– lymphocytes (CD4+CD28– Lc) hold a vital position therein. It is a cell population that combines classical CD4+ lymphocytes’ (helper T cells, Lc Th) characteristics with those of cytotoxic lymphocytes and natural killer cells. Likewise classical Th lymphocytes, they stimulate and control the immunological reaction. However, unlike classical lymphocytes, they participate directly in the reaction, which is expressed by their ability to infiltrate target tissues and possession of cytotoxic properties [15,16,26,31]. CD4+CD28– Lc are resistant to apoptosis, therefore mechanisms induced thereby work incessantly [30]. CD4+CD28– Lc are a source of large quantities of interferon-, a potent macrophage activating factor. Macrophage activation and metalloproteinase excretion is an acknowledged mechanism responsible for the destruction of atherosclerotic plaque fibrous elements, which results in the weakening of the plaque structure and an increased susceptibility to ruptures [18,32]. As a source of granzymes and perforins, the described lymphocytes have an ability to damage the target tissues directly. Cytotoxicity of CD4+CD28– Lc towards endothelial and smooth muscle cells in relation to atherosclerotic plaque was established [15].

It is thought that they may directly damage endothelial cells of intraplaque vessels and consequently lead to the formation of effusions inside a plaque, which undoubtedly contribute to destabilization of atherosclerotic lesions [2,22].

Research on patients with coronary arterial diseases indicates that the described cells play a vital role in the process of coronary artery plaque destabilization and acute coronary syndrome occurrence [11-13].

Our own research has shown so far that the aforesaid cells may also participate in the atherosclerosis-related ischaemic stroke [19]. However, the potential role of the cells in ischemic stroke pathogenesis remains unclear. It seems as if we may seek one in the development of atherosclerotic plaques in extracerebral and intracerebral vessels [6].

The aim of the study was to attempt to address the question of the potential role of CD4+CD28– lymphocytes in the pathogenesis of the atherosclerosis-related ischemic stroke, with special reference to the histopathological picture of carotid atherosclerotic plaques.

Material and methods

The study included 91 patients (25 females and 66 males) aged between 44 and 85 (mean age 67.74), who underwent endarterectomy of internal carotid arteries, with the following comorbidities: arterial hypertension, type 2 diabetes, dyslipidemia, obliterative atherosclerosis of the lower limbs, coronary arterial disease (occurring both separately and in combination with others).

Patients with chronic diseases, where inflammatory and immunological factors play their role, were excluded from the study (systemic connective tissue diseases, viral hepatitis, cirrhosis, ulcerative colitis, Crohn’s disease, multiple sclerosis, Hashimoto’s thyroiditis, Graves’ disease, proliferative diseases of the haematopoietic system, other cancers). Another exclusion criterion was a past surgical procedure performed on a patient within six months directly preceding enrolment in the study.



Microscopic evaluation of carotid plaques



Intraoperatively harvested atherosclerotic plaques were fixed in an 8% formalin solution to be split into five parts. The two most peripherally located parts and one central part, including the area affected by the disease the most, were collected for evaluation. The material was then submerged in paraffin, sliced into 3 micrometer-thick fragments and stained with hematoxylin and eosin (H&E) and using the PAS and Gieson’s methods.

The following were included in the assessment: inflammatory infiltrations, plaque vascularisation, presence of intraplaque haemorrhage, mural thrombus, thrombus built into the plaque structure, plaque fibrous component, foam cells, lipid core, cholesterol crystals and calcifications.

A plaque with a mural thrombosis, fibrous cap rupture and also containing rich lipid core (> 1/3 of the plaque thickness) and/or intraplaque haemorrhage and/or thrombi built into the plaque structure, massive or widespread inflammatory infiltrations (with > 200 or between 100 and 200 cells, respectively), numerous vessels (> 9 vessel sections in the visual field), numerous foam cells (occupying > 1/3 of the plaque thickness), a fibrous component with a majority of collagen fibres (> 2/3) or one with an equal proportion of collagen and elastin fibres, was considered microscopically unstable.

A plaque having no mural thrombi or fibrous cap ruptures but that had intraplaque haemorrhages, intraplaque thrombi or characteristic of at least four out of following features: cholesterol crystals, massive or widespread inflammatory infiltrations, numerous vessels, numerous foam cells, the above described structure of a fibrous component, was considered potentially unstable.

A plaque with no mural thrombosis, no rupture of the fibrous cap, no intraplaque haemorrhages or thrombosis, with minor or large isolated vessels focus (< 6 or between 9 and 6 of the vessel lumen in the visual field), with isolated minor inflammatory infiltrations (< 100 cells in the visual field), scarce foam cells (< 1/3 of the plaque thickness), with neither of the remaining features described above; with no marked disturbance of fibrous element integrity, with a majority of elastin fibres constituting more than 2/3 of the plaque fibrous component, was pronounced as stable.

Plaque calcifications were assessed as numerous (> 8 minor calcifications in the visual field or one large focus occupying more than 1/3 of the plaque thickness), a few minor (between 4 and 8), and isolated minor ones (< 4 focuses in the visual field).

The evaluation was performed on the basis of the American Heart Association Guidelines and an analysis of data available through professional literature, with a particular attention paid to data regarding carotid arteries [2,8,23,29].

Plaques in the ipsilateral artery up to an acute cerebral episode (ischemic stroke or TIA) were regarded as symptomatic. Plaques in patients with no stroke or in the contralateral artery up to a vascular episode were treated as asymptomatic. 46 patients were included in the symptomatic plaque group, while the remaining 45 patients were incorporated into the asymptomatic plaque group.



Flow cytometry



Prior to surgery, each of the subjects included in the study had a 2.7 ml sample of peripheral blood collected to an EDTA tube.

The following antibodies were added to the properly marked tubes (no. 1 and no. 2): to tube no. 1 – control Mouse IgG1-PerCP (Becton Dickinson) and IgG1-PE (Becton Dickinson) antibodies; to tube no. 2 – CD4-PerCP (Becton Dickinson) antibodies and CD28-PE (Becton Dickinson) antibodies. Then, 50 µl of full blood was added to each of the tubes with antibodies. The blood-antibody mix was stored in dark, at room temperature. Post incubation permeabilizing liquid (Dako) was added to samples no. 1 and no. 2. After remixing and incubating, the samples were washed with 5% phosphate buffer saline (PBS). After centrifugation and removing the supernatant, fixing liquid was added to a pellet and to tubes no. 1 and no. 2; the following antibodies were added respectively: Mouse IgG2b-FITC model antibodies (Becton Dickinson) and Anti-human Perforin-FITC (Becton Dickinson). After storing again, washing with a 5% PBS, centrifugation and removing the supernatant, 1% formalin solution was added to a pellet. The expression of the investigated CD28 receptor on lymphocytes and the expression of perforins in the studied cells were evaluated with the application of a FACSCalibur flow cytometer coupled with a sorting device. The analysis was performed on a research computer using Cell Quest OS2 software. Lymphocyte subpopulation was differentiated among lysed whole blood cells, basing on correlated measurements of Forward- and Side-Scattered light (FSC and SSC) (Fig. 1) [21].

The number of the studied CD4+CD28– lymphocytes was expressed as a percentage of CD4+ lymphocytes (CD4+CD28– and CD4+CD28+). The cytotoxicity of the studied cells was assessed on the basis of intracellular perforin expression and was presented as a percentage of CD4+CD28– lymphocytes (Perforin+CD4+CD28– and Perforin-CD4+CD28–) (Fig. 2). Control Mouse IgG1-PerCP (Becton Dickinson) and IgG1-PE (Becton Dickinson) antibodies and Mouse IgG2b-FITC model antibodies (Becton Dickinson) were used as a negative control to exclude non-specific staining (Fig. 3).

The study was conducted upon the approval issued by the Bioethical Commission (BN resolution no. 001/36/06). Informed consent has been obtained from each subject.



Statistical analysis



At the analysis of measurable variables, the following ones were presented: median (Me), minimum value (Min), maximum value (Max) and standard deviation (SD). The measurable variables showed distributions significantly departing from normal distribution (Shapiro-Wilk test, p < 0.05), which is why non-parametric tests were used. To show the significance of differences among more than two groups, ANOVA Kruskal-Wallis was used, and to compare two groups of patients, the Mann-Whitney U test. Nominal variables were compared with the use of the 2 test or its modifications with Yates’s correction or the precise two-sided Fisher’s test (for tables 2 × 2). For a one-factor and then multi-factor analysis of an odds ratio (OR) with 95% confidence interval (95% CI), logistic regression was applied. As a statistical significance threshold, p < 0.05 was assumed. Statistical calculations were made with the use of the Statistica 7.1 programme.

Results

Demographical characteristics of the studied patients, with reference to the symptoms of the operated lesions, are set out in Table I.



Percentage of CD4+CD28– lymphocytes in peripheral blood and their cytotoxicity vs. symptomatic nature of the plaques (plaque ipsilateral to the stroke)



No significant difference was found between the symptomatic plaque group and the asymptomatic plaque one when a global proportion of CD4+CD28– Lc and a proportion of cytotoxic lymphocytes were considered (Table II).



Percentage of CD4+CD28– lymphocytes in peripheral blood and their cytotoxicity vs. instability of atherosclerotic plaques



In the course of a microscopic examination, 54 of the evaluated atherosclerotic lesions met the instability criteria set. In 18 cases, plaques were qualified as potentially unstable. The nature of the remaining 19 plaques was stable (Fig. 4). No significant relation between the proportion of CD4+CD28– Lc and their cytotoxicity and the nature of the studied plaques was observed (Table III).



Percentage of CD4+CD28– lymphocytes in peripheral blood and their cytotoxicity vs. individual histopathological parameters of the plaques (Tables IV and V)



a) CD4+CD28– Lc vs. mural thrombus

The proportion of CD4+CD28– Lc did not constitute a feature that would distinguish patients with plaques complicated by a mural thrombus or lack thereof.

The cytotoxicity of the cells in the former group was higher; however, the difference was not significant.



b) CD4+CD28– Lc vs. intraplaque haemorrhage

Intraplaque haemorrhages were observed in 15 cases. The percentage of CD4+CD28– lymphocytes in peripheral blood of such patients as well as the cytotoxicity of the lymphocytes were comparable to those observed in patients without haemorrhages.



c) CD4+CD28– Lc vs. inflammatory infiltrations

No significant difference in the proportion of the lymphocytes and their cytotoxicity depending on the size of inflammatory infiltrations in the investigated plaques was found.



d) CD4+CD28– Lc vs. plaque vessels

Patients with numerous vessels within atherosclerotic plaques were characterised by a higher percentage of CD4+CD28– Lc when compared to other patients (6.06% vs. 2.57%); nevertheless, the difference was not pronounced. The percentage of cytotoxic lymphocytes was also comparable.



e) CD4+CD28– Lc vs. foam cells

The percentage of lymphocytes, including the percentage of cytotoxic cells, was comparable in patients with both a high and low share of foam cells in the plaque structure.



f) CD4+CD28– Lc vs. plaque fibrous component

In the course of plaque fibrous component analysis, a significantly higher global percentage of the lymphocytes under research was found both in the group of patients with plaques containing mainly collagen fibres and in patients with plaques that were composed of equal proportions of collagen and elastin fibres.

Lymphocyte cytotoxicity was found to have no effect.



g) CD4+CD28– Lc vs. presence of cholesterol crystals

The percentage of the studied cells in peripheral blood was comparable in patients with and without cholesterol crystals present in the plaques. On the other hand, the lymphocyte cytotoxicity was significantly higher in patients with cholesterol crystals.



h) CD4+CD28– Lc vs. intraplaque calcification

No relation between the percentage of lymphocytes in the blood and the degree of calcification of the analysed plaques was observed. However, the percentage of cytotoxic lymphocytes was significantly higher in patients with lower plaque calcification.

A multifactor regression analysis of a dependent variable presented a strong relation between the cytotoxicity of CD4+CD28– lymphocytes and the presence of cholesterol crystals in the plaque (p = 0.0178). No relationship was established in the case of the remaining histopathological plaque parameters.

Discussion

The percentage of CD4+CD28– lymphocytes is increased in the blood of patients with an acute atherosclerosis-related ischemic stroke [19]. Earlier studies stated that their presence in the blood is not a consequence of a stroke. Their high percentage is comparable to that observed in the no-stroke patients with atherogenic risk factors. In both groups, the proportion of the studied lymphocytes in the blood is significantly higher than that of a healthy population [19]. Current results yield similar conclusions. Therefore, the role of CD4+CD28– Lc in ischemic stroke pathogenesis has not been ruled out. They still need to be treated as a factor promoting the development of an acute focal ischemia in the brain.

The presented material included patients with an advanced carotid arteries atheromatosis.

The percentage of CD4+CD28– Lc in the blood was equally high regardless of the type of the analysed risk factors for stroke [20]. These results validate our previous observations and justify the search for potential relations between lymphocytes and atherosclerosis of extracranial arteries [6].

CD4+CD28– Lc, especially their cytotoxic forms, promote atherosclerotic plaque destabilization [12,15]. It seems that these cells in themselves may lead to the stroke (i.e. to symptomatic plaques) [5,22]. In our study. the percentage of CD4+CD28– Lc and their cytotoxicity were comparable in patients with symptomatic and asymptomatic plaques. Therefore, analysed cells are not prognostic factors for stroke (i.e. for symptomatic lesions within carotid arteries).

Research, including our previous studies, also shows that the symptomatic nature of carotid atherosclerotic plaques is rather an outcome of a combined effect of numerous, both intra- and extravascular factors [10,14]. CD4+CD28– Lc could have been only one of such factors by their involvement in the process of plaque destabilization.

A relation of aforesaid circulated lymphocytes to less plaques’ calcification may be an argument for that [17,24,28].

The results indicate a strong relation between the proportion of cytotoxic CD4+CD28– Lc in the blood and the presence of cholesterol crystals in the plaques. Cholesterol crystals within atherosclerotic lesions are recognized as a potent factor promoting inflammation in the wall of an arterial vessel, which results in a cascade of events leading to plaque destabilization [1,4,25]. Other researchers suggest that CD4+CD28– Lc appear originally in the blood and then infiltrate tissues, including atheromatous lesions, in which they enhance inflammatory mechanisms [27].

Therefore, one may suspect that cytotoxic forms of CD4+CD28– Lc indirectly favour plaque destabilization by inflammatory process enhancement, maybe as a reaction to the intraplaque pro-inflammatory cholesterol deposits. However, this thesis needs additional research.

An identification of these cells directly inside carotid plaques may provide more information on the participation of CD4+CD28– Lc in the stroke pathogenesis.

Conclusions

CD4+CD28– cytotoxic lymphocytes seem to be involved in the development of carotid atherosclerotic plaques.

Intraplaque cholesterol deposits may contribute to this process.

References

 1. Abela GS. Cholesterol crystals piercing the arterial plaque and intima trigger local and systemic inflammation. J Clin Lipidol 2010; 4: 156-164.

 2. Alsheikh-Ali AA, Kitsios GD, Balk EM, Lau J, Ip S. The vulnerable atherosclerotic plaque: scope of the literature. Ann Intern Med 2010; 153: 387-395.

 3. Becton, Dickinson and Comapany. Introduction to Flow Cytometry: A Learning Guide. Manual Part Number 11-11032-01 April, 2000, BD Biosciences, pp. 13-14.

 4. Chen Z, Ichetovkin M, Kurtz M, Zycband E, Kawka D, Woods J, He X, Plump AS, Hailman E. Cholesterol in human atherosclerotic plaque is a marker for underlying disease state and plaque vulnerability. Lipids Health Dis 2010; 9: 61.

 5. Executive Committee for the Asymptomatic Carotid Atherosclerosis Study (ACAS). Endarterectomy for asymptomatic carotid artery stenosis. JAMA 1995; 273: 1421.

 6. Gerli R, Schillaci G, Giordano A, Bocci EB, Bistoni O, Vaudo G, Marchesi S, Pirro M, Ragni F, Shoenfeld Y, Mannarino E. CD4+CD28- T lymphocytes contribute to early atherosclerotic damage in rheumatoid arthritis patients. Circulation 2004; 109: 2744-2748.

 7. Golledge J, Greenhalgh RM, Davies AH. The symptomatic carotid plaque. Stroke 2000; 31: 774-781.

 8. Grogan JK, Shaalan WE, Cheng H, Gewertz B, Desai T, Schwarze G, Glagov S, Lozanski L, Griffin A, Castilla M, Bassiouny HS. B-mode ultrasonographic characterization of carotid atherosclerotic plaques in symptomatic and asymptomatic patients. J Vasc Surg 2005; 42: 435-441.

 9. Grymajło-Gójska A, Nyka WM, Zieliński M, Jakubowski Z. CD34/CXCR4 stem cell dynamics in acute stroke patients. Folia Neuropathol 2012; 50: 140-146.

10. Hermus L, Lefrandt JD, Tio RA, Breek J-C, Zeebregts CJ. Carotid plaque formation and serum biomarkers. Atherosclerosis 2010; 213: 21-29.

11. Liuzzo G, Kopecky SL, Frye RL, O’Fallon WM, Maseri A, Goronzy JJ, Weyand CM. Perturbation of T-cell repertoire in patients with unstable angina. Circulation 1999; 100: 2135-2139.

12. Liuzzo G, Goronzy JJ, Yang H, Kopecky SL, Holmes DR, Frye RL, Weyand CM. Monoclonal T-cell proliferation and plaque instability in acute coronary syndromes. Circulation 2000; 101: 2883-2888.

13. Liuzzo G, Biasucci LM, Trotta G, Brugaletta S, Prinnelli M, Digianuario G, Rizzello V, Rebuzzi AG, Rumi C, Maseri A, Crea F. Unusual CD4+CD28null T lymphocytes and reccurance of acute coronary events. J Am Coll Cardiol 2007; 50: 1450-1458.

14. Lutgens E, Suylen R-J, Faber BC, Gijbels MJ, Eurlings PM, Bijnens A-P, Cleutjens KB, Heeneman S, Daemen MJAP. Atherosclerotic plaque rupture: local or systemic process? Arterioscler Thromb Vasc Biol 2003; 23: 2123-2130.

15. Nakajima T, Schulte S, Warrington KJ, Kopecky SL, Frye RL, Goronzy JJ, Weyand CM. T-cell mediated lysis of endothelial cells in acute coronary syndromes. Circulation 2002; 105: 570-575.

16. Namekawa T, Wagner UG, Goronzy JJ, Weyand CM. Functional subset of CD4 T-cells in rheumatoid synovitis. Arthritis Rheum 1998; 41: 2108-2116.

17. Nandalur KR, Baskurt E, Hagspiel KD, Philips CD, Kramer CM. Calcified carotid atherosclerosis plaque is associated less with ischemic symptoms than is noncalcified plaque on MDCT. AJR 2005; 184: 295-298.

18. Newby AC. Metalloproteinases and vulnerable atherosclerotic plaques. Trends Cardiovasc Med 2007; 17: 253-258.

19. Nowik M, Nowacki P, Grabarek J, Drechsler H, Białecka M, Widecka K, Stankiewicz J, Safranow K. Can we talk about CD4+CD28- lymphocytes as a risk factor for ischemic stroke? Eur Neurol 2007; 58: 26-33.

20. Paciaroni M, Bogousslavsky J. Primary and secondary prevention of ischemic stroke. Eur Neurol 2010; 63: 267-278.

21. Pradeep H, Diya JB, Shashikumar S, Rajanikant GK. Oxidative stress – assassin behind the ischemic stroke. Folia Neuropathol 2012; 30: 219-230.

22. Redgrave J-E, Lovett JK, Gallagher PJ, Rothwell PM. Histological assessment of 526 symptomatic carotid plaques in relation to the nature and timing of ischemic symptoms. Circulation 2006; 113: 2320-2328.

23. Redgrave JN, Gallagher P, Lovett J, Rothwell PM. Critical cap thickness and rupture in symptomatic carotid plaques. The Oxford Plaque Study. Stroke 2008; 39: 1722-1729.

24. Rerkasem K, Gallagher PJ, Grimble RF, Calder PC, Shearman CP. The relationship between carotid plaque calcification and stability. Thai J Surg 2011; 32: 131-136.

25. Riazy M, Chen JH, Steinbrecher UP. VEGF secretion by macrophages is stimulated by lipid and protein components of OX-LDL via PI3-kinase and PKCzeta activation and is independent of OXLDL uptake. Atherosclerosis 2009; 204: 47-54.

26. Sawai H, Park YW, Roberson J, Imai T, Goronzy JJ, Weyand CM. T cell costimulation by fractalkine-expressing synoviocytes in rheumatoid arthritis. Arthritis Rheum 2005; 52: 1392-1401.

27. Schmidt D, Goronzy JJ, Weyand CM. CD4+ CD7- CD28- T cells are expanded in rheumatoid arthritis and are characterized by autoreactivity. J Clin Invest 1996; 97: 2027-2037.

28. Shaalan WE, Cheng H, Gewertz B, MCKinsey JF, Schwartz LB, Katz D, Cao D, Desai T, Glagov S, Bassiouny HS. Degree of carotid plaque calcification in relation to symptomatic outcome and plaque inflammation. J Vasc Surg 2004; 40: 262-269.

29. Stary HC, Chandler AB, Dinsmore RE, Fuster V, Glagov S, Insull W, Rosenfeld ME, Schwartz CE, Wagner WD, Wissler RW. A definition of advanced types of atherosclerotic lesions and a histological classification of atherosclerosis: a report from Committee on Vascular Lesions of the Council on Atherosclerosis, American Heart Association. Circulation 1995; 92: 1355-1374.

30. Vallejo AN, Schrimer M, Weyand CM, Goronzy JJ. Clonality and longevity of CD4+CD28null T cells. J Immunol 2002; 168: 3839-3846.

31. Warrington KJ, Takemura S, Goronzy JJ, Weyand CM. CD4+CD28- T cells in rheumatoid arthritis patients combine features of the innate and adaptive immune systems. Arthritis Rheum 2001; 44: 13-20.

32. Weyand CM, Brandes JC, Schmidt D, Fulbright JW, Goronzy JJ. Functional properties of CD4+CD28- T cells in the aging immune system. Mech Ageing Dev 1998; 102: 131-147.