Hindawi Publishing Corporation
Clinical and Developmental Immunology
Volume 2012, Article ID 295081, 8 pages
EnhancedHMGB1 ExpressionMay Contributeto Th17 Cells
1Department of Immunology, Institute of Laboratory Medicine, Jiangsu University, Xuefu Road 301, Zhenjiang 212013, China
2Suzhou Municipal Hospital, Suzhou 215002, China
3The Affiliated People’s Hospital of Jiangsu University, Zhenjiang 212002, China
Correspondence should be addressed to Zhaoliang Su, email@example.com and Huaxi Xu, firstname.lastname@example.org
Received 3 May 2011; Revised 5 July 2011; Accepted 8 July 2011
Academic Editor: Zoltan Szekanecz
Copyright © 2012 Yan Shi et al. This is an open access article distributed under the Creative Commons Attribution License, which
permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Rheumatoid arthritis(RA)is a common autoimmune disease associated with Th17 cells, butwhat about the effect ofhigh-mobility
group box chromosomal protein 1 (HMGB1) and the relationship between Th17-associated factors and HMGB1 in RA remains
unknown. In the present study, we investigated the mRNA levels of HMGB1, RORγt, and IL-17 in peripheral blood mononuclear
cells (PBMCs) from patients with rheumatoid arthritis by quantitative real-time PCR (RT-qPCR), and the concentrations of
HMGB1, IL-17, and IL-23 in plasma were detected by ELISA. And then, the effect of HMGB1 on Th17 cells differentiation was
analyzed in vitro. Our clinical studies showed that the mRNAs of HMGB1, RORγt, and IL-17 in patients were higher than that in
health control (P < 0.05), especially in active RA patients (P < 0.05). The plasma HMGB1, IL-17, and IL-23 in RA patients were
also higher than that in health control (P < 0.05); there was a positive correlation between the expression levels of HMGB1 and
the amount of CRP, ERS, and RF in plasma. In vitro, the IL-17-produced CD4+T cells were increased with 100ng/mL rHMGB1
for 12h, which indicated that the increased HMGB1 might contribute to Th17 cells activation in RA patients.
Rheumatoid arthritis (RA) is an autoimmune disease char-
acterized by chronic inflammation in the small joints leading
to the destruction of articular cartilage and bone. TNF-
α, IL-1, and IL-17 as well as T, B lymphocytes and ma-
crophages are implicated in the pathogenesis of RA [1–3].
Recently, high-mobility group box chromosomal protein 1
(HMGB1), a nonhistone nuclear DNA-binding protein, is
proved to be a potent proinflammatory mediator in rheu-
matoid arthritis [4–7]. Increased HMGB1 was found in the
joints of RA patients [8–10], and the HMGB1 transferred
into health mouse joint could induce the arthritis .
HMGB1 is secreted or released from lymphocytes, dead
and/or apoptosis cells [12–15]. Previous studies have showed
that HMGB1 in milieu could contribute proinflammatory
such as IL-6, IL-1β, and IL-10 secretion by macrophages,
sustain inflammation . It is clear that IL-6 and IL-1β can
prime the na¨ ıve CD4+T cells differentiation into Th17 cells
Whether HMGB1 involved in the pathogenesis of RA
by promoting the Th17 cells activation was unclear. In the
present study, we examined the expression levels of HMGB1
and Th17-associated factors in RA patients, analyzed the
relationship between them, and explored the potentiality of
HMGB1 in Th17 differentiation in vitro.
2.1. Patients. 80 patients with RA enrolled in the affiliated
hospital of Jangsu University were included in this study
from January 2008 to September 2009. Among 80 patients,
59 females and 21 males, ranged from 36 to 80 years old.
48 patients were in active phase, and 32 patients were in
inactive phase. Diagnoses were established according to the
American College of Rheumatology (ACR) criteria  and
2Clinical and Developmental Immunology
the disease activity score calculated for 28 joints (DAS28). 48
patients in active phase untreament during the past 2 years
which did not accompany other chronic diseases; all the 48
patients included 8 males and 40 females, the age was 43 ±
range of DAS28 was 4.21–6.32 (the mean was 5.60 ± 0.78);
the RA patients in inactive phase were 32 cases, 6 males and
26 females, the age was 49 ± 13 years, the course of disease
was 40.1 ± 25.7 months, and the DAS28 range was 1.92 to
0.67 (the average was 1.75 ± 0.23). 50 healthy volunteers, 38
females and 12 males ranged from 28 to 41 years old, acted as
control. This study was approved by the ethical committee of
the Affiliated Hospital of Jiangsu University. All individuals
were informed consensus.
2.2. Reagent. rHMGB1 was expressed in Escherichia coli
(E.coli) and purified by Ni-column. The control protein
eGFP was produced from E.coli and purified by the same
2.3. Blood Samples. Peripheral blood samples were collected
from healthy volunteers and patients. The collection tubes
contained 0.2mL sodium heparin. The blood samples were
centrifugalized at 1000r/min 4◦C for 5min, then the super-
natant was collected and stored at −70◦C for use, and sed-
iment were separated from PBMCs by standard Ficoll-
Hypaque density centrifugation. TRIzol was added to the
PBMCs for total RNA.
2.4. Primers Design. According to Genbank sequences, the
primers were designed by Premier 5.0 software and synthe-
and Service Company. All sequences of primers were shown
in Table 1.
2.5. RNA Extraction and cDNA Synthesis. Following the
manufacturer’s instructions, total RNA from PBMCs was
extracted with Trizol (Invitrogen, USA). cDNA was syn-
thesised with reverse transcription reagent kits (TOYOBO,
Japan). All RNA samples were heated at 65◦C for 10min to
denature the secondary structure with the template then put
in ice for 5min. Total RNA (1μg) was reversely transcribed
in a total volume of 20μL, containing Oligo (dT) 1μL, dNTP
(10mM) 2μL, 5 × RT buffer 4μL, ReverTraAce (100U/μL)
1μL, RNase Inhibitor 1μL, DEPC free H2O add up to 20μL,
response conditions: 42◦C for 20min; 99◦C for 5min; 4◦C
for 5min. The cDNA was stored at −20◦C.
2.6. Construction of Recombinant Plasmid Calibrator. PCR
amplification wasperformed in the Thermon Hybaid System
(Eppendorf, USA). The program consisted of an initial
denaturation step for 5 min at 94◦C followed by 30 cycles,
with each cycle consisting of a 30s denaturing step at 94◦C,
a 30s annealing at 56◦C and a 30s extension at 72◦C. The
reaction was completed by a final 5min extension at 72◦C.
Purified HMGB1, RORγt, IL-17, and β-actin PCR fragments
were transformed to PMD18-T vector (Invitrogen, USA) to
establish recombinant plasmids PMD18-HMGB1, RORγt,
IL-17, and β-actin. All these recombinant plasmids were
transformed into competent E. coli DH5α, transferred on a
1.5% agar Amp-resistant plate, and then cultured at 37◦C
for 12 ∼ 14h. Positive clones were initially identified by se-
quencing. Part of positive clones were further amplified and
extracted and accurately quantified with a nucleic acid-
protein ultraviolet instrument. 10-fold serial dilution of the
recombinant plasmid DNAs were used as calibrator and
stored at −20◦C until use.
2.7. RT-qPCR-Detected Objective Genes Expression. The ob-
jective genes expression (HMGB1, RORγt, and IL-17) were
detected by quantitative real-time polymerase chain reaction
(RT-qPCR), and all samples were calibrated by β-actin.
All PCR reactions were performed using the Rotor-Gene
6000 System (Corbett Research, Australia) in a total volume
of 20μL, containing 1μL cDNA, 10μL 2 × sybr1 premix
(Takara, China), 0.3μL 10μM each primer, and 8.4μL water.
The specificity of the amplification products was controlled
using a melting curve analysis. The copy number of RORγt,
IL-17, and β-actin transcripts in samples was calculated with
the Corbett software according to corresponding standard
curves. The copy number of gene/%β-actin represented the
ratio of the gene. A no-template negative control was also
included in each experiment, and all samples were measured
CD4+T cells from C57BL/6mice spleen were prepared by
magnetic column; 1 × 106/well cells were put into precoating
24-well plates by anti-CD3, anti-CD28, and cultured in
PRIM-1640 including 10% FCS at 5% CO2, 37◦C, the cells
were stimulated with different dose of rHMGB1. The eGFP
0, 3, 6, 9, 12, 24, and 48 hours and supernatants were used to
detect the related cytokines as previously described.
2.9. Enzyme-Linked Immunosorbent Assays (ELISAs) for IL-
17 and IL-23. The levels of HMGB1, IL-17, and IL-23
in plasma or cell culture supernatants were measured by
USA). All samples were measured in triplicate.
2.10. Flow Cytometry Analysis. The procedures of flow cy-
Briefly, 1 × 106PBMCs were stained with anti-CD3-PE-cy5
PE (eBioscience). 1 × 106CD4+T cells from the spleen of
mice were stained with anti-IL-17-FITC (eBioscience). The
stained cells were applied for data acquisition on Coulter
EPICS XL Cytometer (Beckman Coulter) and analyzed by
software WinMDI (version 2.9).
2.11. Statistical Analysis. All statistical analysis were per-
formed using SPSS17.0 statistical analysis software. Data are
expressed as the mean ± standard deviation (SD) in text and
figures. Comparisons between paired or unpaired groups
were performed using the appropriate Student’s t-test. For
Clinical and Developmental Immunology3
Table 1: The primers used in this study.
Gene Sequence(5?–3?) Length (bp)
nonparametric data, differences between two groups were
analyzed by the Mann-Whitney test. Spearman’s correlation
was used to test correlation between two continuous vari-
ables. P < 0.05 was considered to be statistically significant.
3.1. Electrophoresis Identification the Amplicons. The ampli-
con length of HMGB1, RORγt, IL-17, and β-actin was 233,
171, 231, and 265bp, respectively, and it was consistent with
the expected data. The positive clone recombinant plasmid
was identified by sequencing. These objective gene sequences
were in accordance with Genbank seqence (detailed data not
3.2. The Linear Range and Reproducibility. The detection
ies, and the coefficients of variation values ranged from
2.20% to 8.32%. Amplification efficiency ranged from 0.88
to 0.92, and r2> 0.99.
3.3. Levels of HMGB1, RORγt, and IL-17 mRNA in RA
Patients. The expression levels of HMGB1, RORγt, and IL-
17 mRNA from RA patients and healthy controls were
measured by RT-qPCR. As shown in Figure 1, the mRNAs
of Th17-associated cytokines and transcription factor were
significantly increased in PBMCs from RA patients, espe-
from that in inactive phase of patients and healthy controls
(P < 0.05).
3.4. The Correlations between the mRNA Levels of HMGB1
and Th-17 Cells-Related Factors. To assess the relationships
between the mRNA levels of HMGB1 and Th-17 cells-related
factors in RA patients. We examined the correlation between
the mRNA levels of HMGB1 and Th17 cells-related factors
in PBMCs of RA patients. There was a significantly positive
correlation among them (Figure 2).
3.5. Increased Cytokine Concentrations in Plasma from
Patients with RA. Concentrations of plasma HMGB1, IL-23,
and IL-17 measured by ELISA in each group are shown in
increased in plasma from active phase of RA patients, but no
obvious difference between inactive RA and healthy controls.
Genes relative (fold)
Figure 1: Expression of gene ratio by RT-qPCR. The mRNA
expression level were determined by RT-qPCR, the values were
expressed as the fold of the healthy control. the ratio of target genes
used the healthy control as 1.∗P<0.05 compared with the healthy
control and inactive RA group.
The correlations analysis among HMGB1, IL-23, IL-17, and
other clinical targets in the serum of active RA patients
showed that there was a significantly positive correlation
3.6. Increased Frequencies of CD3+CD8−IL-17+T Cells in
PBMCs from RA Patients. Flow cytometry was used to
assess frequencies of CD3+CD8−IL17+T cells in PBMCs
from patients and controls, and the results showed that
CD3+CD8−IL17+T cells in active phase of patients (1.36 ±
0.98%) were higher than those in controls (0.39 ± 0.16%),
and the difference was statistical significance (P < 0.05),
whereas there was no significant difference was found be-
tween inactive phase of patients (0.45 ± 0.23%) and controls
3.7. The mRNA Expression Levels of Th17 Cell-Related Factors
in rHMGB1-Stimulated Mice CD4+T In Vitro. To further
confirm the relationship between HMGB1 and Th17 cells,
4 Clinical and Developmental Immunology
Table 2: The plasma concentration of HMGB1, IL-23, and IL-17 in RA patients.
8.420 ± 1.780∗
203.825 ± 99.321∗
409.239 ± 152.324∗
6.315 ± 0.725
148.332 ± 91.278
188.325 ± 76.143
5.892 ± 0.901
103.825 ± 73.427
165.672 ± 46.238
∗P < 0.05 compared with healthy control.
RORγt relative expression (%)
HMGB1 mRNA expression level (%)
r = 0.451
P = 0.001
00.02 0.040.06 0.08
r = 0.427
P = 0.002
IL-17 mRNA expression level (%)
RORγt mRNA expression level (%)
0 0.02 0.040.060.08
HMGB1 mRNA expression level (%)
0 0.020.04 0.060.08
IL-17 mRNA expression level (%)
r = 0.385
P = 0.007
Figure 2: Correlations of IL-17 and RORγt and HMGB 1mRNA level in RA active patients. The mRNA expression levels as determined by
RT-qPCR, the values were expressed as the target genes versus β-actin mRNA expression.
to detect the mRNA levels of Th17 cell-associated factors by
RT-qPCR. Our data indicated that rHMGB1 could enhance
the expression levels of Th17 cell-related factors, and the lev-
els changed with the dose and time stimulated by rHMGB1.
In the 0.1–100ng/mL rHMGB1 stimulus dose range, Th17-
related factors expression was a dose dependence, 100ng/mL
was the best concentration. Cells were collected at the dif-
ferent points after rHMGB1 stimulation, and Th17 cell-re-
lated factors was up to the peak at 12h (Figure 4).
3.8. CD4+IL-17+T Cells Ratio Was Increased under the
rHMGB1 Stimulation. Flow cytometry analysis showed that
the ratio of IL-17-producing cells (Th17) was up to the max-
for 12h, it shown significant difference compared with other
groups (P < 0.05) (Figure 5).
Th17 cells and their specific transcription factor or related
cytokines are being recognized as important mediators
in inflammatory and autoimmune diseases including RA,
but relatively little is known about HMGB1 roles and the
relationship between Th17 and HMGB1 in RA. In the
present study, we found that in RA patients, the mRNAs
of HMGB1, RORγt, and IL-17 in PBMCs and the levels of
HMGB1, IL-17, and IL-23 in plasma were increased, and
therewasa positive correlationbetween HMGB1-and Th17-
cell, especially in active phase of RA. Furthering analysis
showed that HMGB1 and Th17 related factors also had the
positive correlation with other RA clinical related detections.
To study the relationship of HMGB1 and Th17 cell, we
observed that rHMGB1 could enhance the ratio of Th17 cell.
Clinical and Developmental Immunology5
Figure 3: FACS analyzed the CD3+CD8−IL-17+cell ratio in RA patient The PBMCs were isolated by standard Ficoll-Hypaque density
centrifugation. The cells were stained by anti-CD3-PE-cy5, anti-CD8-FITC, and anti-IL-17-PE. (a) First figure presented CD3+CD8−T cells
were considered CD4+T cells in region RL, and the other three were presented healthy controls. (b) Representative IL-17 expression in
CD3+CD8−T subsets from RA patients in inactive phase. (c) Representative IL-17 expression in CD3+CD8−T subsets from RA patients in
active phase. (d) The results were shown as means ± SD.∗P < 0.05 compared with the control group.
Neither the same source protein nor the endotoxin LPS had
via the Th17 pathway in RA pathogenesis.
There are two pathway of HMGB1 transit from intra-
cellular to extracellular, one is secreted by activated innate
immune cells, the other is released by the death or apoptosis
cells [13–16]. HMGB1 in milieu was involved in the innate
and adaptive immune system . Previous data showed
that HMGB1 could prime the na¨ ıve CD4+T lymphocytes
toward T helper 1 phenotype. Increasing evidence indicated
6 Clinical and Developmental Immunology
Table 3: Correlations of HMGB1, IL-23, IL-17, and clinical index in the serum of active RA patients.
HMGB1 (pg/mL) IL-23 (pg/mL)IL-17 (pg/mL)
∗P < 0.05,#P < 0.01 compared with healthy control.
RORγt induction (fold)
Med u i m
12h 24h48h 72h
IL-17 induction (fold)
Med u i m
RORγt induction (fold)
Med u i m
IL-17 induction (fold)
Med u i m
Figure 4: Th17 cell-related factors expression stimulated by HMGB 1 in vitro. Th17 cell-related factors expression by HMGB 1 stimulus
in vitro were detected by RT-qPCR. The CD4+T cells were isolated from mouse spleen, preactivated by anti-CD3 and anti-CD28, and then
added to rHMGB 1. (a) RORγt mRNA expression levels after rHMGB1 stimulating in different time; (b) IL-17 mRNA expression leves
after rHMGB 1 stimulus; (c) RORγt mRNA expression levels after rHMGB 1 stimulus; (d) IL-17 mRNA expression levels after CD4+T cells
stimulated by rHMGB 1 stimulus. RT-qPCR analysis for target genes versus β-actin mRNA expression, the ratios of target genes used the
control as 1. Data from 3 independent experiments were presented as means ± s.d.∗P < 0.05 versus control.
that HMGB1 acts as an early inflammatory mediator in the
pathogenesis of arthritis [11, 19].
Th17 cells and their effector cytokines are being recog-
nized as important factors in organ-specific autoimmune
diseases, especially which were thought mediating by Th1
cells before [20–25]. Th17 cells have emerged as critical ef-
fector cells in EAE pathogenesis [20, 21]. HMGB1 is a potent
inducer of several proinflammatory cytokines, such as IL-
1β and IL-6, which were considered as crucial mediators in
inducing of Th17 cells . Recently, Liu reported that
HMGB1 can induce IL-23 through TLR4 pathway and IL-23
can enhance the IL-17 levels . Philippa indicates that IL-
23/IL-17 axis exist in the pathogenesis of RA . HMGB1
also played important roles in other autoimmune diseases as
well as in acute allograft rejection [29–32].
In the present study, we not only confirmed the pre-
vious results, but also indicated that the HMGB1 involved
in pathogenesis of RA. There is a positive correlations
Clinical and Developmental Immunology7
100ng/mL 12h 100ng/mL 24h
CD4+IL-17+T cell ratio
Figure 5: The number of IL-17-expressed CD4+T cell stimulated by HMGB 1. The conditions were designed as before. The cells were
collected; before 6h, 1μL monosion, 5μL 10ng/mL PMA, and 1μL 1Mm/mL inon were added. (a) Presents medium control, stimulated
for 12h; (b) Presents independence protein, stimulated for 12h; (c) Presents LPS, stimulated for 12h; (d) Presents 100ng/mL HMGB 1
stimulated for 12h; (e) Presents 100ng/mL HMGB 1 stimulated for 24h; (f) Presents 1000ng/mL HMGB 1 stimulated for 12h.
between HMGB1 and Th17 or other clinical index. Our
data from FACS also showed that HMGB1 might upregulate
CD3+CD8−IL-17+T cells in RA patients, and also in our in
T cells to enhance IL-17 production following activation by
CD3 and CD28 mAbs, which was consistent with our recent
report . In brief, our data provide a strong association
between increased Th17 activity and HMGB1 in RA, and
HMGB1 may upregulate Th17 cells in vivo or in vitro, which
This work was supported by the National Natural Sci-
ence Foundation of China (nos. 30871193, 30972748, and
81001319), the Natural Science Foundation of Colleges and
Universities in Jiangsu Province and Innovation Fund
for candidate of doctor in Jiangsu Province (Grant no.
09KJB310001 and CX09B 217Z, respectively).
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