Introduction
Venous thromboembolism (VTE) includes PE thrombosis
(DVT) and pulmonary embolism (PE) (1) and PE comprises acute pulmonary
embolism (APE) and chronic thromboembolic pulmonary hypertension
(CTEPH). VTE may be acquired or inherited, but is most commonly
acquired, and its prophylaxis is not satisfactory. PE, with its
high incidence, high mortality and high rates of missed diagnosis
and misdiagnosis, has become a major health problem worldwide
(2). The immune system comprises
innate and adaptive defenses and the latter involves cell-mediated
and humoral immune components. We have previously reported that
patients with CTEPH had reduced T cell-mediated immune function
(3) and we have also demonstrated
that VTE is a systemic disease, the incidence of which is related
to viral infection (4).
Interleukins play an important role in immune regulation. Th1
cells, which produce IL2 and IFN-γ, chiefly mediate cellular immune
responses, while Th2 cells, which secrete IL4, IL6 and IL10,
chiefly mediate humoral immune responses (5). In the current study, human cDNA
microarray analysis was used to detect the expression of
interleukin and IFN-γ genes in PBMCs and changes in the acquired
immune functions of PE patients and the control group.
Patients and methods
Patients
A total of 20 patients with PE were recruited from
the Tongji Hospital of Tongji University from 2007 to 2008. A
diagnosis of PE required any two of the following three criteria:
i) selectivity pulmonary angiography shows pulmonary artery
obstruction or filling defect; ii) lung ventilation/perfusion scan
shows single or multiple blood perfusion defect, normal or abnormal
ventilation and V/Q does not match; iii) clinical diagnosis: risk
factors for PE are present and other cardiovascular diseases may be
excluded by clinical performance, electrocardiogram and chest film;
arterial blood gas analysis suggests hypoxemia and hypocapnia; and
D-dimer detection, echocardiography and/or chest computed
tomography support PE diagnosis. We selected 20 patients admitted
to our Department of Cardiology over the same time period to act as
controls. The patients were divided into two groups: i) a PE
patient group comprising 20 patients (11 males and 9 females) with
a mean age of 70±14 (44–89) years, including 3 cases of CTEPH; and
ii) a control group comprising 20 patients (11 males and 9 females)
without PE, DVT, arterial thrombosis or congenital coagulation
abnormality, with a mean age of 72±14 (44–91) years, which were
matched by gender and age with the PE group. No statistically
significant differences were observed between the ages of the two
groups (P>0.05). The clinical trial was approved by the Ethics
Committee of Tongji University and all participants provided their
written informed consent.
Gene expression profiling
Agilent G4112A Whole Human Genome Oligo microarrays
were purchased from Agilent Technologies (Palo Alto, CA, USA). The
microarray comprises 44,290 spots, including 41,675 genes or
transcripts, 314 negative control spots, 1,924 positive control
spots and 359 blank spots. The functions of >70% of genes in the
microarray are known. All patients were subjected to microarray
analysis.
Total RNA isolation
Peripheral blood samples (5 ml) anti-coagulated with
EDTA were drawn from the patients suspected of having PE
immediately after admittance to the hospital and from the patients
without PE. Mononuclear cells were obtained through density
gradient centrifugation with Ficoll solution and the remaining red
blood cells were destroyed with erythrocyte lysis buffer (Qiagen,
Hilden, Germany). Total mononuclear cell RNA was extracted with
TRIzol (Invitrogen, Carlsbad, CA, USA) and purified using a Qiagen
RNeasy column (Qiagen) according to the manufacturer’s
instructions. The isolated total RNA was identified and quantified
using a Nanodrop ND-1000 spectrophotometer (Nanodrop Technology,
Cambridge, UK).
Detection of gene expression
Total RNA (~1 μg) was reverse-transcribed into
double-stranded cDNA. After purification, in vitro
amplification was performed using the Agilent low RNA input linear
amplification kit (Agilent Technologies) and modified UTP [aaUTP,
5-(3-aminoally1)-UTP] was used to replace UTP. The integrated aaUTP
may interact with Cy3 NHS ester to form fluorescent products which
are then used for hybridization. The integration rate of
fluorescence was determined using the Nanodrop ND-1000
spectrophotometer. A hybridization mixture was prepared using an
Agilent oligonucleotide microarray In situ Hybridization
Plus kit. The fluorescent products (~750 ng) were fragmented at
60°C and hybridization was conducted on Human Whole-Genome 60-mer
oligo chips (G4112F, Agilent Technologies) at 60°C for 17 h at 10
rpm. After hybridization, the chips were washed with Agilent Gene
Expression wash buffer according to the manufacturer’s
instructions. Original signals were obtained with Agilent scanner
and Feature Extraction software. The standardization of the
original signals was carried out by the RMA standardized method and
standardized signal values were used for the screening of
differentially expressed genes.
RT-PCR
Three genes, CCL3L3, ZNF683 and LBA1, were selected
from the microarray and their expression was confirmed by RT-PCR.
From the differentially expressed genes, 3 genes were randomly
selected and subjected to RT-PCR together with the housekeeping
gene (GAPDH). The relative expression levels were expressed as the
expression levels of the target genes normalized to the expression
level of GAPDH (2−ΔΔCt). Melting curve and
2−ΔΔCt methods were used to compare the differences in
the expression levels between the control and PE groups. The
results from the RT-PCR procedure were consistent with those from
the microarray analysis.
Statistical analysis
Agilent Feature Extraction software was used to
collect original data from the microarray which was then analyzed
using the robust multichip average (RMA) algorithm. Gene intensity
data for the PE and control groups were compared using the random
variance model corrected t-test. Differentially expressed genes
were identified from whole genomes. P<0.05 was considered to
indicate a statistically significant result.
Results
Differential expression of
interleukins
A total of 37 interleukin genes were detected
(Table I). In comparison with the
control, the expression levels of 12 genes were downregulated
(Figs. 1–3), specifically IL1A, IL9, IL17B, IL19,
IL23A, IL25 (P<0.05), IL2, IL3, IL13, IL22, IL24 and IL31
(P<0.01), and those of 2 of the genes were upregulated,
specifically IL10 and IL28A (P<0.05), in the PE patients.
 | Table IInterleukin gene expression in the
pulmonary embolism (PE) and control groups. |
Table I
Interleukin gene expression in the
pulmonary embolism (PE) and control groups.
| Gene | PE group | Control group |
|---|
| IL1A | 4.47±0.52a | 4.98±0.75 |
| IL1B | 14.79±0.86 | 14.56±0.69 |
| IL2 | 4.47±0.38b | 4.99±0.71 |
| IL3 | 4.37±0.53b | 5.10±0.99 |
| IL4 | 6.39±1.32 | 6.92±1.51 |
| IL5 | 5.10±0.96 | 4.86±0.71 |
| IL6 | 6.66±0.71 | 6.55±0.47 |
| IL7 | 8.40±0.93 | 8.62±0.60 |
| IL8 | 11.27±1.86 | 12.17±1.62 |
| IL9 | 4.51±0.53a | 5.20±1.11 |
| IL10 | 7.68±0.72b | 6.69±0.48 |
| IL11 | 5.11±0.80 | 5.29±0.80 |
| IL12A | 7.03±0.74 | 7.36±0.53 |
| IL12B | 5.46±1.48 | 5.39±0.94 |
| IL13 | 4.58±0.55b | 5.44±1.09 |
| IL15 | 12.21±0.56 | 12.37±0.43 |
| IL16 | 10.71±0.47 | 10.62±0.31 |
| IL17A | 7.02±0.37 | 7.01±0.81 |
| IL17B | 4.33±0.37a | 4.99±1.27 |
| IL17C | 6.07±0.70 | 6.03±0.68 |
| IL17D | 4.90±0.98 | 5.19±0.78 |
| IL17F | 5.78±1.02 | 5.87±1.18 |
| IL18 | 11.02±0.53 | 10.79±0.46 |
| IL19 | 4.90±0.46a | 5.42±0.91 |
| IL20 | 4.78±0.85 | 5.22±0.76 |
| IL21 | 5.56±0.47 | 5.64±0.78 |
| IL22 | 4.18±0.42b | 4.78±0.79 |
| IL23A | 10.68±0.88a | 11.19±0.59 |
| IL24 | 5.15±0.35b | 5.53±0.37 |
| IL25 | 4.62±0.50a | 5.26±0.93 |
| IL26 | 5.37±0.63 | 5.78±1.05 |
| IL27 | 12.26±1.04 | 12.13±1.03 |
| IL28A | 9.38±0.63a | 8.84±0.71 |
| IL28B | 5.00±1.23 | 5.65±1.76 |
| IL29 | 7.05±0.74 | 7.45±0.57 |
| IL31 | 4.18±0.44b | 4.77±0.72 |
| IL32 | 15.06±0.65 | 15.20±0.61 |
mRNA expression of Th1 and Th2
cytokines
The mRNA expression levels of the Th1-type cytokines
IFN-γ and IL2 in the PE patients were significantly lower than
those in the control group (P<0.01), while the mRNA expression
levels of the Th2-type cytokine IL10 were significantly increased
(P<0.01; Tables II and
III).
| Table IIExpression of Th1-type cytokine genes
in the pulmonary embolism (PE) and control groups. |
Table II
Expression of Th1-type cytokine genes
in the pulmonary embolism (PE) and control groups.
| Group | IFN-γ | IL2 |
|---|
| PE group | 9.45±0.94a | 4.47±0.38a |
| Control group | 10.21±0.76 | 4.99±0.71 |
| Table IIIExpression of Th2-type cytokine genes
in the pulmonary embolism (PE) and control groups. |
Table III
Expression of Th2-type cytokine genes
in the pulmonary embolism (PE) and control groups.
| Group | IL4 | IL6 | IL10 |
|---|
| PE group | 6.39±1.32 | 6.66±0.71 | 7.68±0.72a |
| Control group | 6.92±1.51 | 6.55±0.47 | 6.69±0.48 |
Discussion
Human cDNA microarray analysis was used to detect
the expression of interleukin and IFN-γ genes in PBMCs from the PE
and control groups. The differential expression of interleukins and
the Th1/Th2 balance were analyzed. The cytokines included IL2 and
IFN-γ as Th1-type cytokines and IL4, IL6, IL10 as Th2-type
cytokines.
Th precursor cells may be differentiated into Th1 or
Th2 cells. The direction of differentiation is significantly
influenced by a variety of factors, including the cytokine
environment, altered peptide ligands as antigens, varying
quantities of antigen and altered co-stimulatory signals (6). Th1 lymphocytes secrete IL2, IFN-γ and
IL12, which regulate cell-mediated immunity by promoting the
differentiation, maturation and proliferation of T lymphocytes and
increasing the activity of NK cells. Th2 lymphocytes secrete IL4,
IL6 and IL10 and promote the differentiation, maturation and
proliferation of B lymphocytes and the generation of antibodies.
Changes in the levels of Th1 and Th2 cytokines reflect the immune
balance. A shift of the Th1/Th2 balance may cause an immunological
regulation function disorder.
The immune system needs to maintain a balance since
imbalance will cause it to over- or under-respond. In the adaptive
immune system, cell-mediated immunity and humoral immunity also
require balance and this is primarily regulated by Th1 and Th2
cytokines.
In the current study, the mRNA expression levels of
the Th1 cytokines IFN-γ and IL2 in the PE patients were
significantly downregulated (P<0.01), while the mRNA expression
levels of the Th2 cytokine IL10 were significantly upregulated,
suggesting reduced Th1 activity and enhanced Th2 activity. The
shift of the Th1/Th2 balance indicates a disorder of the
immunological regulation function. Downing et al have
reported elevated IL10 in the vein wall during venous thrombosis
(7) and elevated levels of IL6
have been reported in DVT (8).
These studies show increased levels of Th2 cytokines and
enhancement of the humoral immune response in VTE, which supports
our results.
We have previously reported that the numbers of
CD3+ and CD8+ T cells in patients with CTEPH
are significantly reduced (3) and
that virus-like structures were synthesized and released in a
patient with pulmonary hypertension (9). We have also previously found that T
cell and NK cell immunity function-associated genes were
downregulated in patients with PE (10), and that CD3+
CD8+ T cell immunity is dysfunctional in acute PE
patients (11). These studies
demonstrate a compromised cell-mediated immunity. In the current
study, a shift of the Th1/Th2 balance along with enhanced Th2
activity in PE patients was demonstrated, indicating that
cell-mediated immunity was compromised. The result is consistent
with the reduced T cell function reported in the aforementioned
studies
Our findings demonstrate the association between
symptomatic VTE and adaptive immunity. The imbalance of Th1/Th2
manifests as reduced cell-mediated immunity and increased humoral
immunity, which indicates that the immune system plays a role in
the regulation of the immune balance. The upregulation of humoral
immunity may be the result of the regulation of the immune system
to establish a new balance. The immune system comprises two major
parts, the innate immune system and the adaptive immune system,
which defend against invading organisms. The function of T cells
and NK cells in cell-mediated immunity is mainly antiviral, while
the principal function of B cells in humoral immunity is the
generation of antibodies. When there is a disturbance in immune
function, the ratio of Th1/Th2 shifts and the body is susceptible
to infection. Infection is a risk factor for VTE (12), therefore individuals who are
vulnerable to infection may be susceptible to VTE.
Acknowledgements
This study was supported by the National Natural
Science Foundation of China (30871080).
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