Int. J. Med. Sci. 2017, Vol.

14 181

Ivyspring
International Publisher International Journal of Medical Sciences
2017; 14(2): 181-190. doi: 10.7150/ijms.17119
Research Paper

Immune Cell Repertoire and Their Mediators in Patients

with Acute Myocardial Infarction or Stable Angina
Pectoris
Wenwen Yan1, Yanli Song2, Lin Zhou1, Jinfa Jiang1, Fang Yang3, Qianglin Duan1, Lin Che1, Yuqin Shen1,
Haoming Song1, Lemin Wang1
1. Department of Cardiology, Tongji Hospital, Tongji University School of Medicine, Shanghai 200065, China;
2. Department of Emergency Medicine, Tongji Hospital, Tongji University School of Medicine, Shanghai 200065, China;
3. Department of Experimental Diagnosis, Tongji Hospital, Tongji University School of Medicine, Shanghai 200065, China.

Corresponding authors: Lemin Wang, Haoming Song, Yuqin Shen, Department of Cardiology, Tongji Hospital, Tongji University School of Medicine, 389
Xincun Rd, Putuo District, Shanghai 200065, China; Tel: 86 21 66111329, Fax: 86 21 66111329, E-mail: wanglemin@tongji.edu.cn; songhao-ming@163.com;
sy-1963@126.com.

Ivyspring International Publisher. This is an open access article distributed under the terms of the Creative Commons Attribution (CC BY-NC) license
(https://creativecommons.org/licenses/by-nc/4.0/). See http://ivyspring.com/terms for full terms and conditions.

Received: 2016.08.05; Accepted: 2016.12.21; Published: 2017.02.08

Abstract
Background: To evaluate the natural innate and adaptive immunity through gene expression and
cytology levels in peripheral blood mononuclear cells in patients with acute myocardial infarction
(AMI), stable angina pectoris (SAP) and controls.
Methods: 210 patients with AMI, 210 with SAP, and 250 clinical controls were recruited. Whole
human genome microarray analysis was performed in 20 randomly chosen subjects per group
were examined to detect the expressions of complement markers, natural killer cells, T cells and
B cells. The quantity of these cells and related cytokines as well as immunoglobulin levels were
measured in all subjects.
Results: In AMI group, the mRNA expressions of late complement component, markers of
natural killer cells, CD3+, CD8+ T cells and B cells were down-regulated, while those of early
complement component and CD4+T cells were up-regulated (p<0.05). In both AMI and SAP
patients, the quantity of natural killer cells, CD3+, CD8+ T cells, B cells, IgM and IgG were
significantly lower than those of the controls. CD4+ T cells, CH50, C3, C4, IL-2, IL-4, IL-6 and
IFN- were significantly higher (p<0.05).
Conclusions: In AMI patients, both of gene expressions related to complement, natural killer
cells, CD3+, CD8+ T cells, B cells and the quantity of these immune cells decreased while cell
number reduced in SAP patients. Immune function in both AMI and SAP patients decreased
especially in AMI patients with declined gene and protein levels. To improve the immune system is
a potential target for medical interventions and prevention in AMI.
Key words: myocardial infarction, stable angina pectoris, gene expression, innate immunity, adaptive
immunity.

Introduction
Cardiovascular diseases (CADs), with high and prevent AMI occurrence. The pathologic
morbidity and mortality worldwide, are caused mechanism responsible for majority of AMI is the
mainly by atherosclerosis. In particular, acute rupture of stable atherosclerotic plaque and
myocardial infarction (AMI) represents life- thrombosis [3]. Obviously, there must be a trigger to
threatening conditions during the history of CAD [1, induce the sudden rupture. Infection seems to be
2]. Nowadays we are still unable to effectively predict undoubtedly linked to vulnerable atherosclerotic

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lesion; however, its role cannot be easily documented the following: 1) Symptoms of ischemia. 2) New or
[4, 5, 6]. Various exogenous microorganism infections, presumed new significant ST-segment-T wave (ST-T)
including Chlamydia pneumoniae, Helicobacter changes or new left bundle branch block (LBBB). 3)
pylori, Cytomegalovirus and Bacteroides gingivalis Development of pathological Q waves in ECG. 4)
are accepted as the new susceptible factors of CAD [7, Imaging evidence of new loss of viable myocardium
8]. or new regional wall motion abnormality.5)
Our recent study demonstrated the decreased T Identification of an intracoronary thrombus by
cell immunity function in AMI patients [9, 10]. T cells, angiography.
as a key component of adaptive immune system, All SAP patients had exclusively effort-related
eliminate the pathogenic microorganisms and angina with a positive exercise stress test and at least
malignant cells. The significant decline of T cell one coronary stenosis was detected at angiography
function suggests that the pathogenesis of acute (>70% reduction of lumen diameter).
thrombosis in AMI patients may be associated with There were no significant differences among
the depletion of immune cells. However, less is three groups in age, sex, body mass index (BMI),
known about the nature of immune response in ethnicity, smoking status, systolic blood pressure
different stages of CAD [11, 12]. In recent study, we (SBP), diastolic blood pressure (DBP), low-density
designed this in vitro study to investigate both innate lipoprotein cholesterol (LDL-C), triglycerides,
and adaptive immunity in patients with AMI or stable high-density lipoprotein cholesterol (HDL-C) and
angina pectoris (SAP). Human microarray analysis fasting plasma glucose (FBG) (Table 1).
was used to systematically measure them RNA The exclusion criteria for three groups were as
expression of the complement component, markers of follows: venous thrombosis, history of severe renal or
immune cells in peripheral blood mononuclear cells hepatic diseases, hematological disorders, acute or
(PBMCs) from AMI, SAP and controls. Moreover, the chronic inflammatory diseases and malignancy.
quantity of immune cells, related cytokines and The study protocol was approved by the ethics
immunoglobulin levels were also measured. committee of Tongji University and informed consent
form was obtained.
Material and Methods
Table 1. Baseline demographic data in three groups ( x s.d.).
Patients Information
Index AMI (a) SAP (b) Con(c) P P
The study recruited 210 patients with AMI, 210 (N=210) (N=210) (N=250) (all) (a v b)
with SAP, and 250 clinically controls. Human Age 58.510.7 63.611.1 60.9 9.4 0.141 0.211
microarray analysis was performed in 20 randomly Sex(M/F) 180/30 176/34 207/43 0.694 0.773
BMI(kg/m2) 24.62.9 22.52.2 22.71.9 0.112 0.76
chosen subjects per group. The sample sizes and the
Ethnicity, Han 210 210 250 1 1
number of subjects per group were based on an Tobacco 13.610.1 14.48.4 11.26.1 0.24 0.648
assumed within-group variance of 0.50 and the smoking(num/d)
targeted nominal power of 0.95 [13]. Table 1 shows the SBP (mmHg) 130.111.3 123.710.1 124.87.8 0.145 0.701
DBP (mmHg) 67.78.8 72.08.8 77.63.6 0.126 0.24
baseline demographic data. All patients were enrolled LDL-C(mmol/L) 2.81.2 2.41.8 2.71.5 0.44 0.676
between Mar 2013 and Feb 2015 from our Coronary Triglycerides(mmol/L) 1.51.8 1.71.0 1.80.7 0.51 0.12
Care Unit and Cardiovascular Department. The AMI HDL-C(mmol/L) 0.70.9 0.80.7 0.90.2 0.11 0.303
FBG (mmol/L) 5.30.4 5.10.7 5.00.2 0.24 0.834
patients were admitted no more than 12 hours from
Footnotes: BMI= body mass index; SBP=systolic blood pressure; DBP =diastolic
the onset of symptoms to our Coronary Care Unit blood pressure; LDL-C=low-density lipoprotein cholesterol; HDL-C: high-density
including 180 males and 30 females, with an average lipoprotein cholesterol; FBG: Fasting Plasma Glucose.

age of 5911 years. The SAP group included 210

patients (176 males, 34 females, aged 6411 years). 250 Gene Expression Chips
healthy volunteers (207 males, 43 females, aged 61 9
Agilent G4112F Whole Human Genome Oligo
years) were enrolled as the control group during the
Microarrays purchased from Agilent (USA) were used
same period. Histories, physical examination, ECG,
in the chip analysis. A microarray is composed of
chest radiography and routine chemical analyses
more than 41,000 genes or transcripts, including
showed the controls had no evidence of coronary
targeted 19,596 entrez gene RNAs. Sequence
heart diseases.
information used in the microarrays was derived from
All AMI patients were diagnosed on the basis of
the latest databases of RefSeq, Goldenpath, Ensembl
following criteria [14]: Detection of a rise of cardiac
and Unigene [15]. More than 70% of the gene
biomarker values [preferably cardiac troponin (cTn)]
functions in the microarray are already known. All 20
with at least one value above the 99th percentile
randomly selected patients for each group were
upper reference limit (URL) and with at least one of
subjected to the chip analysis.

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Total RNA Isolation RT-PCR. Among all the genes with different
Ten milliliter of peripheral blood samples from expressions, three genes were randomly selected and
median cubital vein were drawn from all the patients subjected to RT-PCR, along with the house keeping
immediately after admission. Four milliliter blood genes (GAPDH). The relative expressions were
was kept in PAXgene tube for total RNA isolation and indicated as the expression of the target genes
the rest six milliliter was for laboratory assays. normalized to the expression of GAPDH (2-Ct).
Leucocytes were obtained through density gradient The melting curve and the 2-Ct-method were used
centrifugation with Ficoll solution and the remaining to detect the differences in the expressions among the
red blood cells were destroyed by erythrocyte lysis three groups. The results from RT-PCR were
buffer (Qiagen, Hilden, Germany). Following the consistent with the microarray analysis.
manufacturers instructions, total RNA was extracted Laboratory assays
and purified using PAXgeneTM Blood RNA kit
Two milliliter blood sample was anticoagulated
(Cat#762174, QIAGEN, GmBH, Germany). We further
with EDTA-K3 for the counting of CD16+CD56+
checked for a RIN number to inspect RNA integration
natural killer cells, T lymphocyte subsets and CD19+B
by an Agilent Bio analyzer 2100 (Agilent technologies,
cells, and the rest four milliliter was separated by
Santa Clara, CA, US). The sample was considered
centrifugation within 1 hour for the examination of
qualified when both 2100 RIN and 28S/18S were
serum immunoglobulin and cytokines. All tests were
larger than or equal to 0.7.
finished within two weeks.
RNA Amplification and Labeling CH50 was detected with liposome immune assay
Total RNA was amplified and labeled by Low (Beckmann DxC-800 fully automatic biochemical
Input Quick Amp Labeling Kit, One-Color analyzer, USA; Reagents: Wako Pure Chemical
(Cat#5190-2305, Agilent technologies, Santa Clara, Industries, Ltd., Japan). C3 and C4 were detected with
CA, US), following the manufacturers instructions. immunone-phelometry (BNII system, Siemens AG,
Labeled cRNA was purified by RNeasy mini kit Germany; Reagents: Siemens Healthcare Diagnostics
(Cat#74106, QIAGEN, GmBH, Germany). Products GmbH, Germany).
Cytokines, including IL-2, IL-4, IL-6 and IFN-
Microarray Hybridization were measured by double antibody sandwich ELISA
Each slide was hybridized with 1.65g assay (Microplate reader Model 2010, Anthos,
Cy3-labeled cRNA using Gene Expression Austria; Reagents: Dili biotech, Shanghai). Serum
Hybridization Kit (Cat#5188-5242, Agilent levels of IgA, IgM and IgG were calculated by the
technologies, Santa Clara, CA, US) in Hybridization immunonephelometric technique using the
Oven (Cat#G2545A, Agilent technologies, Santa automated IMMAGE 800 immunochemistry system
Clara, CA, US), following the manufacturers (Beckman Coulter, Brea, CA, USA), and expressed as
instructions. After 17 hours of hybridization, slides g/L.
were washed in staining dishes (Cat#121, Thermo Leukocyte subpopulations were measured by
Shandon, Waltham, MA, US) with Gene Expression flow cytometry (BEPICS XL-4, BECKMAN-
Wash Buffer Kit (Cat#5188-5327, Agilent technologies, COULTER). Monoclonal antibodies against CD3,
Santa Clara, CA, US), according to the manufacturers CD4, CD8, CD16, CD56 and CD19 were purchased
operation manual. from BD Biosciences. The antibodies were marked
with one of three fluorochromes: fluorescein
Chip Scan and Data Acquisition isothiocyanate (FITC), phycoerythrin (PE) and
Slides were scanned using Agilent Microarray phycoerythrin-cyanin 5.1 (PC5). Cells were identified
Scanner (Cat#G2565CA, Agilent technologies, Santa by combinations as follows: CD3 (FITC)/CD16
Clara, CA, US) with default settings. Dye channel: (PE)/CD56 (PC5) (NK cells), CD3 (FITC)/CD4
Green, Scan resolution=3m, 20bit. Data were (PE)/CD8 (PC5) (CD4andCD8 cells), and CD19 (PE)
extracted with Feature Extraction software 10.7 (B cells). In brief, 100 L of EDTA treated blood was
(Agilent technologies, Santa Clara, CA, US). Raw data added to each tube and control tube was also
were normalized using Quantile algorithm, Gene included. 20 L of mouse IgG1-FITC, IgG1-PE or
Spring Software 11.0 (Agilent technologies, Santa IgG1-PC5 was then added, followed by addition of
Clara, CA, US). corresponding fluorescence antibodies. Following
vortexing, incubation was done in dark for 30 min at
RT-PCR
room temperature. 500 L of hemolysin (BECKMAN-
The spots in the microarray were randomly COULTER) was then added, followed by incubation
selected and their expressions were confirmed by at 37C for 30 min. Following washing, 500 L of

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sheath fluid was added to each tube, followed by flow Results

cytometry (EPICS XL-4, BECKMAN-COULTER). The
PMT voltage, fluorescence compensation and Gene expression and serum level of the
sensitivity of standard fluorescent microspheres complement
(EPICS XL-4, BECKMAN-COULTER) were used to The results showed mRNA expressions of early
adjust the flow cytometer and a total of 10,000 cells and late complement components including C1q,
were counted for each tube. The corresponding cell C1q, C1q, C1r, C1s, C2, C3, C4b, C5a, C6, C7, C8,
population in the scatter plot of isotype controls was C8, and C8 and C9 were examined in PBMCs from
used to set the gate, and the proportion of positive three groups of patients (Figure 1A). In AMI group,
cells was determined in each quadrant (%). gene expressions of C1q (p<0.05), C1q, C1q, C1r
SYSTEM-II was used to process the data obtained and C5a were significantly up-regulated (all p<0.01),
after flow cytometry. whereas expressions of C7, C8 and C9 were
significantly down-regulated when compared with
Statistical Analysis
SAP patients and controls, respectively (p<0.05). C1s
Descriptive statistics were expressed as mean expression in AMI patients was significantly lower
s.d. Differences between groups were examined by than the controls (p<0.05). Serum CH50, C3 and C4
one-way analysis of variance (ANOVA). After levels were significantly increased in AMI and SAP
ANOVA the test of all pairwise group mean patients when compared with controls (p<0.01). CH50
comparison was performed using the Tukey's was significantly higher in AMI patients than in SAP
method. Density curves for CH50, C3, C4, patients (p<0.01). There was no significant difference
CD16+CD56+, CD3+, CD4+, CD8+and CD19+ cells between AMI and SAP patients in C3 and C4 levels.
were delineated using R software. Data were The density curves of CH50, C3 and C4 are shown in
analyzed using SPSS 17.0, and p-values <0.05 were Figure 1B-D separately.
considered statistically significant.

Figure 1. From three groups in PBMCs, (A) mRNA expression of early and late components complement. (B) Serum CH50 level. (C) Serum C3 level. (D) Serum C4
level. Three groups: *, P<0.05; **, P<0.01. AMI vs. Con: #, P<0.05; ##, P<0.01. AMI vs. SAP: +, P<0.05; ++, P<0.01. SAP vs. Con &: P<0.05; &&: P<0.01.

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Gene expression and counting of NK cells SAP patients and the controls (p<0.05). There was no
statistical difference in NK cell biomarker expressions
The results showed 12 gene expressions of NK
between SAP and the controls. Density curves for the
cell biomarkers[16], including CD16, CD56, five
NK cell proportion in PBMCs from three groups were
inhibitory receptors,CD94, NKG2A, CD158(KIR2DL),
delineated (Figure 2B). The two density curves of cell
CD161(KLRB1), CD328( Siglect-7) and five activating
proportion from AMI and SAP patients in PBMCs
NK cell receptors, including CD335(Nkp46),
were substantially left shift when compared with the
CD337(Nkp30), CD48(2B4), CD314(NKG2D) and
controls. The number of NK cells was significantly
CD319(CRACC) in PBMCs from three groups (Figure
decreased in both AMI and SAP patients (p<0.01).
2A). In AMI group mRNA expressions of the genes
However, there was no significant difference between
encoding CD94, NKG2A, CD158, CD161, CD337,
AMI and SAP patients in the quantity of NK cells.
CD314 and CD319 were significantly lower than in

Figure 2. From three groups in PBMCs, (A) mRNA expression of intracellular and extracellular markers of CD16+CD56+cells. (B) The comparison of
CD16+CD56+ cells counting. Three groups: *, P<0.05; **, P<0.01. AMI vs. Con: #, P<0.05; ##, P<0.01. AMI vs. SAP: +, P<0.05; ++, P<0.01. SAP vs. Con &: P<0.05;
&&: P<0.01.

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Gene expression, subsets counting and related Between SAP and control group, there was no
cytokines of T cells statistical difference in TCR, CD4+ and CD8+T cell
markers related mRNA expression.
Expressions of 8 genes related to T cell receptor Results from the proportions of cytological T
(TCR) antigen recognition, 16 genes associated with lymphocyte subsets in PBMCs among three groups
CD4+T cells and 15 genes with CD8+ T cells were showed the levels of CD3+ and CD8+T cells in AMI
detected among three groups (Figure 3A, 3C, 3E). 16 and SAP group decreased significantly (p< 0.05),
genes in AMI patients encoding TCRA, TCRB, TCRG, while CD4+T cells increased (p<0.01) when compared
TCRZ, CD3D, CD3E, CD3G, CD195(CCR5), IL-10, with control group (Figure 3B, 3D, 3F, Table 2). The
GATA3, CD278(ICOS), CD8A, CD8B, CD28, GZMM cytokine IL-2, IL-4, IL-6 and IFN were significantly
and CASP10 were significantly down-regulated when increased in AMI and SAP patients when compared
compared with the SAP patients and controls with the controls (p<0.01). However, there was no
respectively(p<0.05). TCRIM, CD294 (CRTH2) and significant difference between AMI and SAP patients
GZMK expressions in AMI group were significantly in IL-2, IFN, CD3+ T cell and CD8+T cell quantity
lower than those in SAP group (p<0.05). Comparing (Table 2). The counting of CD4+ T cell, IL-4 and IL-6
with controls, gene expressions of CD4, IL4 and were higher in AMI patients than in SAP patients
TNFA in AMI group were significantly down- (p<0.01).
regulated (p<0.05), while IL-2 and CD366 (Tim-3)
mRNA expressions were up-regulated (p<0.05).

Figure 3. From three groups in PBMCs, (A) Expression of genes related to T cell antigen recognition. (B) CD3+ counting. (C) Expression of genes related to CD4+.
(D) CD4+ counting. (E) Genes related to CD8+. (F) CD8+ counting. Three groups: *, P<0.05; **, P<0.01. AMI vs. Con: #, P<0.05; ##, P<0.01. AMI vs. SAP: +, P<0.05;
++, P<0.01. SAP vs. Con &: P<0.05; &&: P<0.01.

Table 2. Values of T cell immunity among three groups ( x s.d.).

Index AMI (a) SAP (b) Con(c) P P P P
(N=210) (N=210) (N=250) (all) (a/c) (b/c) (a/b)
CD3+ (%) 66.710.7 68.410.0 71.59.2 0.00 0.00 0.002 0.275
CD4+ (%) 42.18.9 45.1.9.2 37.09.1 0.00 0.00 0.00 0.003
CD8+ (%) 21.87.1 23.36.7 28.66.9 0.00 0.00 0.00 0.068
IL-2 (pg/ml) 34.218.5 33.514.5 9.92.3 0.00 0.00 0.00 0.96
IL-4(pg/ml) 35.715.7 28.2210.9 4.82.3 0.00 0.00 0.00 0.00
IL-6 (pg/ml) 29.916.2 24.614.4 3.01.4 0.00 0.00 0.00 0.001
IFN (pg/ml) 40.821.4 33.022.1 16.76.3 0.00 0.00 0.00 0.72

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Gene expression, counting and serum from SAP and control group (p<0.05). Compared with
immunoglobulin level of B cells controls, gene expressions of CD21, CD23 and CD79a
were significantly down-regulated in AMI patients
The results showed that expressions of 15 genes (p<0.05). Between the SAP and control group, there
related to B cell biomarkers in patients with AMI, SAP were no significant differences in B cell marker
and the controls (Figure 4A), including CD5, CD19, expressions. When compared with controls, B cell
CD20, CD21 (CR1), CD22, CD23, CD40, CD79a, counting, IgG and IgM in PBMCs were significantly
CD79b, CD80(B7-1), CD86(B7-2), CD138, CD154(IgM), down-regulated (p<0.01), while IgA was significantly
CD268(BAFFR) and CD279(PD-1). In PBMCs from increased in both AMI and SAP group (Figure 4B,
three groups, expressions of 8 genes encoding CD5, Table 3) (p<0.05).
CD19, CD20, CD22, CD40, CD79b, CD268 and CD279
in AMI group were significantly lower than those

Figure 4. From three groups in PBMCs, (A) mRNA expression of intracellular and extracellular markers of CD19+ cell. (B) The comparison of CD19+ cells counting.
Three groups: *, P<0.05; **, P<0.01. AMI vs. Con: #, P<0.05; ##, P<0.01. AMI vs. SAP: +, P<0.05; ++, P<0.01. SAP vs. Con &: P<0.05; &&: P<0.01.

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Table 3. Values of B cell immunity among three groups ( x s.d.).

Index AMI(a) SAP (b) Con(c) P P P P
(N=210) (N=210) (N=250) (all) (a/c) (b/c) (a/b)
CD19+(%) 9.45.0 10.34.7 13.74.6 0.00 0.00 0.00 0.212
IgA (g/L) 2.31.0 2.20.8 1.90.7 0.00 0.001 0.014 0.487
IgM(g/L) 0.80.42 0.80.35 1.20.41 0.00 0.00 0.00 0.572
IgG (g/L) 10.82.6 11.22.2 12.02.3 0.00 0.00 0.001 0.242

patients with AMI. The cytotoxic ability of NK cells

Discussion was decreased afterwards. There was no significant
In our current study, the significantly difference in mRNA expression between the SAP
up-regulated mRNAs expressions of early patients and controls in inhibitory and activating
complement components, C1q, C1q, C1q, C1r and receptors, indicating the NK receptors in SAP patients
C5a demonstrated that the classical pathways were was in a nearly inactive state. Previous studies found
activated in AMI patients. The initiation of classical the reduced proportions of NK cells in peripheral
pathway eventually results in the terminal access to blood of CAD, but the reason was still controversial
form C5b-9 complex, which makes a transmembrane [28, 29, 30, 31]. The similar loss of NK cell numbers in
pore in the target cells' membrane to lysis [17]. C5b both AMI and SAP patients were also observed in our
initiates the formation of MAC, which consists of C5b, study (Figure 2B). Together with the notably
C6, C7, C8, and multiple molecules of C9. In our study decreased expression of NK cell biomarkers in AMI
the significantly lowest levels of C7, C8 and C9 patients, different levels of reduced immunity in NK
mRNAs in AMI patients suggested the obstacle of cells were demonstrated in AMI and SAP stages. In
MAC formation. In AMI and SAP patients, the serum AMI patients both numbers and receptor activity
levels of C3, C4 and CH50, which reflected the were decreased, while only a deficit of quantity was
activities of C1-C9 via classic pathway, were all found in SAP patients.
elevated. Gene and cytology levels of the complement TCR is a molecule found on the surface of T
in both AMI and SAP patients were activated and the lymphocytes that is responsible for recognizing
results were consistent with previous studies [18, 19, antigens. The first signal for T cell activation is
20]. Though the complement was activated in AMI provided through the TCR-CD3 [32]. In present study,
and SAP stages, based on the genomics results of gene expressions of TCRA, TCRB, TCRG, TCRZ,
complement cascade reaction imbalance, cytolytic CD3D, CD3E and CD3G were significantly lower in
effect of the complement only decreased in AMI AMI group than those in SAP and control group
patients. (Figure 4A), indicating the decreased ability of TCR
NK cells express an array of inhibitory and antigen recognition. In addition, the loss of CD3+ T
activating receptors. The inhibitory receptors are cells in PBMCs was found in both AMI and SAP
responsible for self-tolerance while activating patients (Figure 4B), suggesting the dysfunction of
receptors mediate the NK cell cytotoxicity (NKCC) CD3+T cells in CAD, especially in AMI stage.
[21, 22]. KIRs are the most important NK cell Naive CD4+ T cells differentiate into T helper
receptors, including CD94, NKG2A, CD158 and type 1 (Th1) and T helper type 2 (Th2). Th1 cells
CD314, which recognize classical MHC class I [23]. In achieve cellular immunity mainly by secreting IL2,
present study, the gene expressions of CD94, NKG2A IL12 and IFN-. T-bet is a Th1 transcription factor
and CD158 were significantly lower than those in SAP for regulating Th1 development [33]. CD195 (CCR5)
patients and the controls, suggesting the impaired and CD182 (CXCR3) are specific Th1 lymphocytes
ability to protect normal cells in AMI patients. chemokine receptors [34]. Th2 cells produce IL4, IL6
Receptors CD335 (NKp46), CD337 (NKp30), CD48 and IL10 to activate B lymphocytes and generate
(2B4), CD314(NKG2D) and CD319 (CRACC) are most antibodies. GATA3 is the Th2 specific transcription
central activating receptors and play an important factor, and CCR3 together with CD294 (CRTH2) are
role in targeting NK cell responses toward abnormal chemokine receptors of Th2 cells [35, 36, 37]. CD366
cells and eventually the cell lysis [24, 25, 26, 27]. In our (Tim-3) is a Th1-specific cell surface protein while
current study, gene expressions of activating CD365 (Tim-1) is Th2-specific [38, 39]. The high
receptors, CD337, CD314 and CD319 in AMI patients mRNA expressions of Th1 biomarkers (IL2 and
were significantly decreased in comparison with SAP CD366) and low RNA expressions of Th2 biomarkers
patients and controls respectively, which showed the (IL4, IL-10, CD278 and CD294) in AMI patients
transduction of activating signal was inhibited in suggested a shift towards Th1 dominance. The
significant increase of CD4+T cells, IL-4 and IL-6 in

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AMI than in SAP patients showed the differential of NK cells, CD3+, CD8+ T cells and CD19+ B cells
degrees of CD4+ T cell mediated cellular immunity were decreased, while the gene expression of immune
dysfunction in AMI and SAP patients. system was in a nearly inactive status. In the current
CD8+ T cells kill virus-infected cells and tumor study, we can conclude that the attack of AMI and
cells and play a critical role in immune protection [40]. SAP was associated with different levels of immune
CD8+T cell is firstly activated by TCR and CD8 dysfunction. AMI occurred in the stage of immune
binding and then co-stimulatory molecules.CD8+ T collapse while SAP occurred in progressively reduced
cells make the fatal attack through the level of immunity but still within the boundary of
perforin-granzyme, Fas-Fas ligand (FasL), and TNF-a compensation. The quantity of immune cells in
pathways [41,42]. The presence of CD8+ T cells in peripheral blood may reflect the current state of
atherosclerotic lesions is widely demonstrated but immune function and the gene expression of immune
studies investigating their role in atherogenesis have system stands for the compensatory capacity of the
yielded contradictory results [43, 44]. In the present immune system.
study, all 15 genes related to killing ability of CD8+ T In AMI patients, the suppressed innate and
cells in AMI patients were down-regulated; especially adaptive immune system, especially the cytotoxic
CD8A, CD8B, CD28, CD278, GZMK, GZMM, PRF1 ability, failed to remove the exogenous pathogens.
and CASP10 were significantly down-regulated when Various exogenous microorganism infections are
compared with SAP and/or controls. Together with supposed as risk factors of AMI [8, 9] and infection
significant loss of CD8+T cells in AMI and SAP seems to be linked to plaque rupture [4, 5, 6, 7].
patient in PBMCs indicated the decreased cytotoxic
ability of CD8+ T cells in CAD patients, particularly in Conclusions
the stages of AMI patients. The pathogenesis of AMI might be related with
We detected all 15 genes related with infections of pathogens under the depletion of
intracellular and extracellular markers of CD19+B immune system. That is the reason why single vaccine
cells [45] (Figure 4A). B cell receptor (BCR) was is ineffective on AMI prevention. To improve the
composed of membrane immunoglobulin (Ig) which immunity of CAD patients may be considered as a
recognizes the antigens while Ig (CD79A) and Ig potential target for medical intervention and
(CD79B) transmit the activation signals [46]. CD19 prevention of AMI.
and CD21 are B cell co-receptors and enhance the BCR
signal transduction [47]. The B cell specific Src-family Acknowledgements
kinase CD5, specifically binding B cell surface Ig, is The study was supported by Shanghai
dispensable for B cell activation [48]. CD268 is the Traditional Chinese Medicine 3-year Development
principal receptor required for BAFF-mediated B cell Program (2014-2016); National Natural Science
activation [49]. CD279 encodes a cell surface Foundation (81570359) and Shanghai municipal
membrane protein of Ig superfamily and plays a role health and Family Planning Commission project
in their differentiation [50]. In AMI patients, gene (20144Y0046).
expressions of CD5, CD19, CD20, CD21, CD22, CD23,
CD40, CD79A, CD79B, CD268 and CD279 were Competing Interests
significantly lower than those in SAP and /or control The authors have declared that no competing
group, which showed B cell activation were blocked interest exists.
in AMI patients. There was no significant gene
expression difference in B cell activation between the References
SAP patients and the control group. The detection of B [1] Libby P, Ridker PM, Hansson GK. Progress and challenges in translating the
cell quantity, IgM and IgG levels in PBMCs were biology of atherosclerosis. Nature. 2011; 473: 317325.
[2] Libby P, Theroux P. Pathophysiology of coronary artery disease. Circulation.
decreased in both AMI and SAP patients. In sum, in 2005; 111: 3481-3488.
[3] Shah PK. Pathophysiology of coronary thrombosis: role of plaque rupture and
AMI patients, gene expressions and numbers of B plaque erosion. Prog Cardiovasc Dis. 2002; 44:357-368.
cells were reduced, demonstrating the deeply [4] Levi M, van der Poll T, Schultz M. New insights into pathways that determine
the link between infection and thrombosis. Neth J Med. 2012; 70:114-120.
weakened humoral immunity in AMI group. [5] Chatzidimitriou D, Kirmizis D, Gavriilaki E, et al. Atherosclerosis and
In the present study, in AMI patients the mRNA infection: is the jury still not in? Future Microbiol. 2012; 7:1217-1230.
[6] Levi M, van der Poll T, Schultz M. Infection and inflammation as risk factors
expression of immune system was consistent with the for thrombosis and atherosclerosis. Semin Thromb Hemost. 2012; 38:506-514.
cytological level and the decline of both parameters [7] Khan S, Rahman HN, Okamoto T, et al. Promotion of atherosclerosis by
Helicobacter cinaedi infection that involves macrophage-driven
demonstrated the collapse of immune function in proinflammatory responses. Sci Rep. 2014; 4: 4680.
AMI group. In SAP patients, the immunity related [8] Mostafa A, Mohamed MK, Saeed M, et al. Hepatitis C infection and clearance:
impact on atherosclerosis and cardiometabolic risk factors. Gut. 2010;
gene expression was different from cytological level. 59:1135-1140.
The CH50, C3 and C4 were increased and the number

http://www.medsci.org
Int. J. Med. Sci. 2017, Vol. 14 190
[9] Yan WW, Zhang KS, Duan QL, et al. Significantly reduced function of T cells [43] Kyaw T, Winship A, Tay C, et al. Cytotoxic and proinflammatory CD8+ T
in patients with acute arterial thrombosis. Journal of Geriatric Cardiology. lymphocytes promote development of vulnerable atherosclerotic plaques in
2015; 12: 287-293. apoE-deficient mice. Circulation. 2013; 127:1028-1039
[10] Yan WW, Wang LM, Jiang JF, et al. Differential expression of T cell-related [44] Zhou J, Dimayuga PC, Zhao X, et al. CD8 (+) CD25 (+) T cells reduce
genes in AMI and SA stages of coronary artery disease. Int J ClinExp Med. atherosclerosis in apoE (-/-) mice. Biochem Biophys Res Commun. 2014;
2015; 8:10875-10884. 443:864-870.
[11] Matusik P, Guzik B, Weber C, et al. Do we know enough about the immune [45] Pike KA, Ratcliffe MJ. Cell surface immunoglobulin receptors in B cell
pathogenesis of acute coronary syndromes to improve clinical practice? development. Semin Immunol. 2002; 14:351-358.
Thromb Haemost. 2012; 108:443-456. [46] Kurosaki T. Regulation of BCR signaling. Mol Immunol. 2011; 48:1287-1291.
[12] Arbab-Zadeh A, Nakano M, Virmani R, et al. Acute coronary events. [47] Barrington RA, Schneider TJ, Pitcher LA, et al. Uncoupling CD21 and CD19 of
Circulation. 2012; 125: 11471156. the B-cell coreceptor. Proc Natl Acad Sci USA. 2009; 106:14490-14495.
[13] Dobbin K, Simon R. Sample size determination in microarray experiments for [48] Pospisil R, Silverman GJ, Marti GE, et al. CD5 is A potential selecting ligand
class comparison and prognostic classification. Biostatistics. 2005; 6:27-38. for B-cell surface immunoglobulin: a possible role in maintenance and
[14] Thygesen K, Alpert JS, Jaffe AS, et al. Third universal definition of myocardial selective expansion of normal and malignant B cells. Leuk Lymphoma. 2000;
infarction. J Am Coll Cardiol. 2012; 60: 1581-1598. 36:353-365.
[15] Wiltgen M, Tilz GP. DNA microarray analysis: principles and clinical impact. [49] Bergmann H, Yabas M, Short A, et al. B cell survival, surface BCR and BAFFR
Hematology. 2007; 12:271-287. expression, CD74 metabolism, and CD8- dendritic cells require the
[16] Cooper MA, Colonna M, Yokoyama WM. Hidden talents of natural killers: NK intramembrane endopeptidase SPPL2A. J Exp Med. 2013; 210: 31-40.
cells in innate and adaptive immunity. EMBO Rep. 2009; 10:1103-1110. [50] Thibult ML, Mamessier E, Gertner-Dardenne J, et al. PD-1 is a novel regulator
[17] Lappegrd KT, Garred P, Jonasson L, et al. A vital role for complement in of human B-cell activation. Int Immunol. 2013; 25:129-137.
heart disease. Mol Immunol. 2014; 61:126-134.
[18] Iltumur K, Karabulut A, Toprak G, et al. Complement activation in acute
coronary syndromes. APMIS. 2005; 113: 167-174.
[19] Cusack MR, Marber MS, Lambiase PD, et al. Systemic inflammation in
unstable angina is the result of myocardial necrosis. J Am Coll Cardiol. 2002;
39:19171923
[20] Leinoe E, Pachai A, Brandslund I. Complement activation reaches maximum
during equilibrium between antigen and antibody in an in vitro model for
thrombolysis with streptokinase. APMIS. 2000; 10:685688.
[21] Backstrm E, Kristensson K, Ljunggren HG. Activation of natural killer cells:
underlying molecular mechanisms revealed. Scand J Immunol. 2004; 60: 14-22.
[22] Middleton D, Curran M, Maxwell L. Natural killer cells and their receptors.
Transpl Immunol. 2002; 10: 147-64.
[23] Campbell KS, Purdy AK. Structure/function of human killer cell
immunoglobulin-like receptors: lessons from polymorphisms, evolution,
crystal structures and mutations. Immunology. 2011; 132:315-325.
[24] Raulet DH, Gasser S, Gowen BG, et al. Regulation of ligands for the NKG2D
activating receptor. Annu Rev Immunol. 2013; 31:413-441.
[25] Zafirova B, Wensveen FM, Gulin M, et al. Regulation of immune cell function
and differentiation by the NKG2D receptor. Cell Mol Life Sci. 2011;
68:3519-3529.
[26] Koch J, Steinle A, Watzl C, et al. Activating natural cytotoxicity receptors of
natural killer cells in cancer and infection. Trends Immunol. 2013; 34:182-191.
[27] Claus M, Meinke S, Bhat R, et al. Regulation of NK cell activity by 2B4, NTB-A
and CRACC. Front Biosci. 2008; 13: 956-965.
[28] Whitman SC, Rateri DL, Szilvassy SJ, et al. Depletion of natural killer cell
function decreases atherosclerosis in low-density lipoprotein receptor null
mice. Arterioscler Thromb Vasc Biol. 2004; 24:1049-1054
[29] Li W, Lidebjer C, Yuan XM, et al. NK cell apoptosis in coronary artery disease:
relation to oxidative stress. Atherosclerosis. 2008; 199:65-72.
[30] Jonasson L, Backteman K, Ernerudh J. Loss of natural killer cell activity in
patients with coronary artery disease. Atherosclerosis. 2005; 183:316-321.
[31] Backteman K, Ernerudh J, Jonasson L. Natural killer (NK) cell deficit in
coronary artery disease: no aberrations in phenotype but sustained reduction
of NK cells is associated with low-grade inflammation. Clin Exp Immunol.
2014; 175:104-112.
[32] Zehn D, King C, Bevan MJ, et al. TCR signaling requirements for activating T
cells and for generating memory. Cell Mol Life Sci. 2012; 69: 1565-1575.
[33] Vanaki E, Ataei M, Sanati MH, et al. Expression patterns of Th1/Th2
transcription factors in patients with guttate psoriasis. Acta Microbiol
Immunol Hung. 2013; 60: 163-174.
[34] Gao P, Zhou XY, Yashiro-Ohtani Y, et al. The unique target specificity of a
nonpeptide chemokine receptor antagonist: selective blockade of two Th1
chemokine receptors CCR5 and CXCR3. J Leukoc Biol. 2003; 73: 273-280.
[35] Wan YY. GATA3: a master of many trades in immune regulation. Trends
Immunol. 2014; 35: 233-242.
[36] Sallusto F, Mackay CR, Lanzavecchia A. Selective expression of the eotaxin
receptor CCR3 by human T helper 2 cells. Science. 1997; 277: 2005-2007.
[37] Nagata K, Hirai H. The second PGD< sub> 2</sub> receptor CRTH2:
structure, properties, and functions in leukocytes. Prostaglandins Leukot
Essent Fatty Acids. 2003; 69: 169-177.
[38] Hastings WD, Anderson DE, Kassam N, et al. TIM-3 is expressed on activated
human CD4+ T cells and regulates Th1 and Th17 cytokines. Eur J Immunol.
2009; 39:2492-2501.
[39] Curtiss ML, Gorman JV, Businga TR, et al. Tim-1 regulates Th2 responses in an
airway hypersensitivity model. Eur J Immunol. 2012; 42:651-661.
[40] Gadhamsetty S, Mare AFM, Beltman JB, et al. A General Functional Response
of Cytotoxic T lymphocyte Mediated Killing of Target Cells. Biophys J. 2014;
106: 1780-1791.
[41] Keefe D, Shi L, Feske S, et al. Perforin triggers a plasma membrane-repair
response that facilitates CTL induction of apoptosis. Immunity. 2005; 23:
249-262.
[42] Berke G. The CTL's kiss of death. Cell. 1995; 81: 9-12.

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