Correlation of CD95 and soluble CD95 expression with acute rejection status of liver transplantation.

Abstract

To analyze the expression levels of soluble form of CD95, CD95 ligand (sCD95 and sCD95L, respectively) in plasma and CD95 expression on CD3+ cells in liver-transplanted recipients with acute rejection (AR).
Peripheral blood mononuclear cells (PBMCs) were isolated from 30 clinically liver transplanted recipients. CD95 expression on CD3+ cells was quantitatively measured by two-color fluorescence activated cell sorter (FACS) analysis. Lymphocyte surface phenotypes of CD4, CD8, CD16 and CD56 were determined by flow cytometry. Plasma levels of sCD95 and sCD95L were detected by Enzyme Linked-Immuno-Sorbent Assay (ELISA). The results were compared with that from normal healthy volunteers (n = 15 individuals).
FACS analysis showed that CD95 expression on CD3+ T cells was significantly increased in liver transplanted recipients with AR compared to that in stable recipients without rejection and infection or healthy individuals who did not undergo transplantation (18,676.93+/-11,588.34/molecule, 6,848.20+/-1 712.96/molecule, 6,418.01+/-2,001.95/molecule, respectively, P<0.01). Whereas no significant difference was seen between liver-transplanted stable recipients and healthy individuals. Furthermore, no significant differences were detected between each group with CD4/CD8 ratio or the percentage of CD16+56+ cells. Plasma levels of sCD95 were significantly higher in transplanted recipients with AR compared to that in stable recipients or healthy individuals (391.88+/-196.00, 201.37+/-30.30, 148.83+/-58.25 pg/mL, respectively, P<0.01). In contrast, the plasma levels of sCD95L in liver- transplanted recipients were not significantly different from that in healthy individuals.
The present results indicate that the increased CD95 expression on CD3+ cells and the increased levels of sCD95 in plasma may modify the immunological situation of the recipients after transplantation or represent the ongoing graft rejection.

Full text

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• BRIEF REPORTS •
Correlation of CD95 and soluble CD95 expression with acute
rejection status of liver transplantation
Yu-Liang Wang, Yan-Yan Zhang, Guang Li, Zhi-Qin Tang, Yan-Li Zhou, Zhi-Jun Zhu, Zhi Yao
ELSEVIER
PO Box 2345, Beijing 100023, China World J Gastroenterol 2005;11(11):1700-1704
www.wjgnet.com
wjg@wjgnet.com © 2005 The WJG Press and Elsevier Inc. All rights reserved.
World Journal of Gastroenterology ISSN 1007-9327
Yu-Liang Wang, Zhi-Qin Tang, Tianjin Institute of Thrombosis
and Hemostasis, Laboratory Center, Tianjin First Central Hospital,
24 FuKang Road, Tianjin 300192, China
Yan-Yan Zhang, INSERM U362, Institut Gustave Roussy, PR1,
39 Rue Camille Desmoulins, 94805 Villejuif, France
Guang Li, Department of Biology, Tianjin Medical University,
Tianjin 300070, China
Yan-Li Zhou, Department of Rheumatosis, First Teaching Hospital
Tianjin University of Traditional Chinese Medicin, Tanjin 300193, China
Zhi-Jun Zhu, Organ Transplantation Center, Tianjin First Central
Hospital, 24 FuKang Road, Tianjin 300192, China
Zhi Yao, Department of Immunology, Tianjin Medical University,
Tianjin 300070, China
Correspondence to: Dr. Yu-Liang Wang, Tianjin Institute of
Thrombosis and Hemostasis, Laboratory Center, Tianjin First Central
Hospital, 24 FuKang Road, Tianjin 300192,
China. wang_yu_l@yahoo.com.cn
Telephone: +86-13001335062 Fax: +86-22-23626600
Received: 2004-09-08 Accepted: 2004-11-24
Abstract Abstract
Abstract Abstract
Abstract
AIM: To analyze the expression levels of soluble form of
CD95, CD95 ligand (sCD95 and sCD95L, respectively) in
plasma and CD95 expression on CD3+ cells in liver-
transplanted recipients with acute rejection (AR).
METHODS: Peripheral blood mononuclear cells (PBMCs)
were isolated from 30 clinically liver transplanted recipients.
CD95 expression on CD3+ cells was quantitatively measured
by two-color fluorescence activated cell sorter (FACS)
analysis. Lymphocyte surface phenotypes of CD4, CD8,
CD16 and CD56 were determined by flow cytometry.
Plasma levels of sCD95 and sCD95L were detected by
Enzyme Linked-Immuno-Sorbent Assay (ELISA). The
results were compared with that from normal healthy
volunteers (n = 15 individuals).
RESULTS: FACS analysis showed that CD95 expression on
CD3+ T cells was significantly increased in liver transplanted
recipients with AR compared to that in stable recipients without
rejection and infection or healthy individuals who did not
undergo transplantation (18 676.93±11 588.34/molecule,
6 848.20±1 712.96/molecule, 6 418.01±2 001.95/molecule,
respectively, P<0.01). Whereas no significant difference
was seen between liver-transplanted stable recipients and
healthy individuals. Furthermore, no significant differences
were detected between each group with CD4/CD8 ratio
or the percentage of CD16+56+ cells. Plasma levels of sCD95
were significantly higher in transplanted recipients with AR
compared to that in stable recipients or healthy individuals
(391.88±196.00, 201.37±30.30, 148.83±58.25 pg/mL,
respectively, P<0.01). In contrast, the plasma levels of
sCD95L in liver- transplanted recipients were not significantly
different from that in healthy individuals.
CONCLUSION: The present results indicate that the
increased CD95 expression on CD3+ cells and the increased
levels of sCD95 in plasma may modify the immunological
situation of the recipients after transplantation or represent
the ongoing graft rejection.
© 2005 The WJG Press and Elsevier Inc. All rights reserved.
Key words: Liver transplantation; Acute rejection; CD95
Wang YL, Zhang YY, Li G, Tang ZQ, Zhou YL, Zhu ZJ, Yao
Z. Correlation of CD95 and soluble CD95 expression with
acute rejection status of liver transplantation. World J
Gastroenterol 2005; 11(11): 1700-1704
http://www.wjgnet.com/1007-9327/11/1700.asp
INTRODUCTIONINTRODUCTION
INTRODUCTIONINTRODUCTION
INTRODUCTION
Numerous studies have proposed several mechanisms of
graft rejection in liver transplantation such as the manipulation
of intragraft cytokines, apoptosis of graft infiltrating
lymphocyte and so on. Cytokines play an important role in
regulating immunological responses of the host against
transplanted organs. They control the activation and
differentiation of immune effector cells and mediate
cytotoxic activity of the effector cells. It has been suggested
that Type I CD4+ and CD8+ T cells (Th1, Tc1) cytokines
(interleukin-2, interferon-γ and tumor necrosis factor
(TNF)-α) might promote cellular rejection[1-3], whereas type
II CD4+ and CD8+ T cells (Th2, Tc2) cytokines (interleukin-4
and interleukin-10) might suppress graft rejection[4].
T cells stimulated via the T-cell receptors (TCRs) not
only proliferate, but also undergo subsequent apoptosis by
activation-induced cell death (AICD)[5,6]. Much evidence
indicates the implication of AICD in the immune responses
against alloantigens. Recent studies have been reporting that
some pathways involved AICD of T cells[7,8]. CD95/CD95
ligand (CD95L) pathway has been shown to be a major
AICD mediator of T cells. CD95 (Fas or APO-1) and its
ligand are cell surface proteins. CD95 is a 48-KD type I
transmembrane protein member belonging to the TNF
receptor family, and CD95L is a 40-KD type II integral
membrane protein belonging to the TNF family[9]. The
interaction between the CD95 and CD95L is recognized as
a major pathway for the induction of apoptosis in cells and
tissues[10]. It has been suggested that the CD95 and CD95L

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play an important role in the regulation of immune
responses to foreign antigens and in the induction of
peripheral tolerance[11]. sCD95 has recently been detected
in the serum of the patients with liver diseases, including
injury, hepatitis, cirrhosis, hepatocellular carcinoma (HCC)
and in the systemic lupus erythematosus patients[12,13]. It has
been shown that human CD95L was secreted from activated
T cells[14] and the sCD95L may act as a cytotoxic cytokine,
although Tanaka and colleagues recently suggested its
inhibitory function[15]. CD95/CD95L pathway is regulated
by a number of implicating factors. Till now, the role of
CD95 and CD95 ligand system in graft rejection is not
fully understood and changes of their expression during
liver allograft rejection have not been elucidated.
Given the above considerations, we aimed to examine
CD95 expression by CD3+ T cells and the plasma levels of
sCD95 and sCD95L in human recipients after liver
transplantation, and estimated their relation to the liver
rejection. The significance of these results on liver
transplantation will be discussed below.
MAMA
MAMA
MATERIALS AND METHODS
TERIALS AND METHODS
TERIALS AND METHODSTERIALS AND METHODS
TERIALS AND METHODS
Patient
Thirty liver -transplanted recipients (24 men, 6 women) who
were treated at Organ Transplantation Center of Tianjin
First Central Hospital were divided into post-transplanted
AR (n = 15) and post-transplanted stable (n = 15) groups.
The patients had been transplanted within 3 mo of
immunosuppressed treatment with FK506, zenapax, MMF,
and steroids. AR was diagnosed by means of clinical, laboratory,
and histologic evidence. Methylprednisolone was used for
treatment. Blood samples were collected from AR group
and stable group after liver- transplantation. The control
group consisted of 15 healthy individuals. Peripheral blood
mononuclear cells (PBMCs) were separated by Ficoll-Hypaque
density gradient centrifugation from ethylenediaminetetraacetic
acid (EDTA) blood 5 mL. Plasma was collected from healthy
donors and patients by centrifugation of heparinized
peripheral blood (PB) at 3 000 r/min for 10 min. Plasma
samples were divided into aliquots and stored at -70
until measured.
Methods
Plasma cytokines The concentration of sCD95 in plasma
was determined by a solid phase sandwich Enzyme Linked-
Immuno-Sorbent Assay (ELISA) (DIACLONE France).
Briefly, a monoclonal antibody (mAb) specific for sCD95
has been coated onto the wells of the microliter strips provided.
Plasma and standards of known sCD95 concentrations are
pipetted into these wells. During the first incubation, the
antigen and a biotinylated mAb specific for sCD95 are
simultaneously incubated. After five washes with 0.05%
Tween 20-phosphated buffered saline (PBS), pH7.4, the
enzyme (streptavidin-peroxydase) is added. After incubation
and washing, to remove all unbound enzyme, a substrate
solution, which acts with the bound enzyme, is added to
induce a colored product. The intensity of this colored
product is directly proportional to the concentration of
sCD95 present in the plasma. The same ELISA system for
sCD95L was used for the in vitro quantitative determination
of sCD95L in plasma.
Determination of lymphocyte subpopulations Staining
with mAb was performed in 100 µL aliquots of heparinized
PB, followed by lysis of red blood cells and fixation with 1%
paraformaldehyde (PAF). Phycoerythrin (PE) conjugated
mouse anti-human CD4, CD8, CD16, fluorescein
isothiocyanate (FITC) conjugated mouse anti-human CD56
and isotype-matched control mAbs (All from Becton
Dickinson) were used. 10 000 events were acquired using
fluorescence activated cell sorter (FACS) Calibur and analysis
was performed using CELLQUEST software (Becton
Dickinson).
Quantitative measurement of cell surface expression
of CD95 (Fas/Apo-1) expression on CD3+ lymphocytes
by dual-color flow cytometry Fifty microliter of cell
suspension was added to each of 2-5 mL polystyrene snap
cap tubes. To the first tube (T1), 25 µL negative isotypic
control (BIOCYTEX, France) was added. To the second
tube (T2), 25 µL FITC conjugated mouse anti-human CD95
(Fas/Apo-1) mAb (BIOCYTEX) was added. To the third
tube (T3), 50 µL of QuantiBRITE beads (BDB) suspension
(BIOCYTEX), which were coated with increasing and
accurately known quantities of mouse immunoglobulins G
was added. Samples were incubated for 10 min at room
temperature. Subsequently, in each of the tube, 10 µL of
PE conjugated mouse anti-human CD3 mAb (Becton
Dickinson) was added. Samples were incubated at room
temperature for 10 min, followed by washing in PBS with
2% fetal calf serum and fixation in 1% PAF and then
analyzed by flow cytometry. The standard curve (Figure 1C)
was made by plot the MFI (mean fluorescence index)
calibration values obtained from T3 on the X-axis and their
corresponding number of mAb molecules on the Y-axis
(Figure 1B). Note the MFI values of T1 and T2 obtained
on the corresponding histogram after gating CD3+ cells
(Figure 1A). Interoplate the MFI values of T1 and T2,
then read the corresponding number of mAbs directly off
the curve. The number of specific sites was obtained by
subtracting T1 value to T2 value.
Statistical analysis
All data are presented as the mean±SD. Statistic analyses
were performed with the t-tests. P-values of less than 0.05
were regarded as significant throughout the study.
RESULRESUL
RESULRESUL
RESULTS
TS
TS TS
TS
We analyzed and compared freshly isolated PBMCs from
liver-transplanted patients and healthy individuals as regards
CD95 expression on CD3+ T cell by dual-color flow
cytometry (Figure 1). We did not find any significant
difference between post-transplanted stable recipients and
healthy individuals. However, CD95 expression on CD3+ T
cells was significantly increased in liver transplanted recipients
with AR compared with that in stable recipients without
rejection and infection or healthy individuals who did not
undergo transplantation (P<0.01, Table 1).
We also investigated the sCD95 and sCD95L in plasma
by ELISA. Plasma levels of sCD95 were significantly higher
Wang YL et al. CD95 and soluble CD95 and liver allograft rejection 1701

Page 3

in transplanted recipients with AR than that in stable
recipients or healthy individuals (P<0.01). In contrast, the
levels of sCD95L in liver transplanted recipients were not
significantly different from that expressed in healthy
individuals (Table 1).
As concerns T lymphocyte subpopulations, no significant
differences were seen among each group with CD4/CD8
ratio and the percentage of CD16+56+ cells (Table 2).
Table 1 Levels of CD95 expression on CD3+cells (/molecule) and
levels of sCD95, sCD95L (pg/mL) in plasma from post-transplanted
AR group, post-transplanted stable group and healthy group
Post-transplanted Post-transplanted Healthy group
stable group (n = 15) AR group (n = 15)(n = 15)
CD95 (/ molecule) 6 848.20±1 712.96b 18 676.93±11 588.34 6 418.01±2 001.95d
sCD95 (pg/ mL) 201.37±30.30 b
391.88±196.00 148.83±58.25 d
sCD95L (pg/ mL) 279.42±79.72 226.82±132.63 254.38±96.29
bP<0.01 vs post-transplanted stable group; dP<0.01 vs health group.
Table 2 CD4/CD8 ratio, the percentage of CD16+56+ cells in post-
transplanted AR group, post-transplanted stable group and healthy
group
Post-transplanted
stable group (n = 15) AR group (n = 15)
Post-transplanted Healthy group
(n = 15)
CD4/ CD8 1.5±0.5 1.8±0.4 1.5±0.2
CD16+56+ (%) 15.4±9.5 20.4±9.4 17.0±7.5
DISCUSSION DISCUSSION
DISCUSSION DISCUSSION
DISCUSSION
Acute rejection (AR) is a well-known complication of allograft
transplantation. T lymphocytes appear to be absolutely
required in this rejection process[16,17]. Fas (CD95) is expressed
in resting peripheral blood T cells and the Fas antigen as
well as the ligand (FasL, CD95L) are up-regulated following
T cell activation. Increased expression of FasL may activate
the cytotoxic pathway, leading to the graft damage by Fas
system activation and result in apoptosis.
The studies of Rivero et al., showed that up-regulated
expression of Fas antigen in liver tissue with liver rejection
but not in liver tissue without rejection, suggesting the
importance of the Fas system in the rejection of liver
grafts[18]. Our studies, which focused on the peripheral blood
lymphocytes, showed that CD95 expression on CD3+ cells
did not have any significant difference between liver-
transplanted stable recipients without rejection and infection
and healthy individuals who did not undergo transplantation.
This result was similar to that of Renzo, who demonstrated
that Fas on CD3+ T cell and FasL mRNA expression in
PBMC have no significant difference between cardiac-
transplanted subjects and normal controls[19]. However,
when compared to the recipients with AR to the stable
recipients or healthy individuals, we found that the CD95
expression on CD3+ cells was significantly increased. This
increasing may have been caused by antigen stimulation.
Stimulation through the CD3-TCR complex up-regulates
CD95 expression and induces CD95L expression[20,21].
Through these cell surface molecules, activated T cells can
commit suicide through formation of CD95-CD95L
complexes[22,23]. Therefore, we suppose that because of the
higher CD95 expression, CD3+ T lymphocyte in AR patients
may undergo more apoptosis than that from stable recipients,
so that the spontaneous tolerance to the allograft may
develop. On the other hand, previous animal studies of
cardiac, renal and liver transplantation demonstrated that
FasL up-regulation in allografts in rejection condition[24,25].
Therefore, we cannot exclude that in AR patients, the CD95
higher expression T lymphocyte may infiltrate in the allograft
and induce apoptosis through formation of CD95-CD95L
complex, then accelerate the rejection process.
In the meantime, we determined the plasma levels of
sCD95 in liver- transplantation recipients. The sCD95 results
from the deletion of the transmembrane domain of CD95.
The levels of sCD95 were significantly increased in liver
transplanted recipients with AR compared to that in stable
recipients or healthy individuals. It has been speculated
that sCD95 may reflect the expression levels of Fas antigen
on tissue and the severity of inflammatory activity[26-30].
Therefore, the sCD95 detected in the present samples might
be shedding from injured organs that express CD95. The
significant increase of sCD95 in AR recipients were strong
associated with the serum levels of total bilirubin, AST and
ALT (unpublished data), implying that the levels of sCD95
may reflect the graft damage, which could be useful when
Figure 1 Quantitative measurement of cell surface expression of CD95 expression on CD3+ lymphocytes PBMCs were labeled with QuantiBRITE
beads suspension (T3), CD95-FITC (T2) or isotype control IgG1-FITC (T1) and CD3-PE as described in materials and methods. A: Histogram
showed the expression of CD95 on CD3+ cells. Solid histogram is isotype control. Bold line: CD95; B: Histogram showed the fluorescence
intensity of the mouse immunoglobulins G in T3; C: The standard curve was made by plot the MFI calibration values obtained from Figure 1B
on the X-axis and their corresponding number of mAb molecules on the Y-axis.
Counts
100 101 102 103 104
0
200
160
120
80
40
A
CD95-FITC
Counts
100 101 102 103 104
80
70
60
50
40
30
20
10
0
B
FL1-H
MAb molecules
0.1 1.0 10 100 1 000 10 000
1 000 000
100 000
10 000
1 000
100
C
MFI (a.u.)
M1
M2
M3M4
1702 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol March 21, 2005 Volume 11 Number 11

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evaluating response to treatment. A previous study
demonstrated the expression of CD95L mRNA in kidney-
transplanted grafts at AR[31]. However, in this study, plasma
levels of sCD95L in liver- transplanted recipients were not
significantly different from that in healthy individuals,
indicating that sCD95L might not contribute to AR.
Concomitant immunosuppression, which is routinely
administered in human transplantation cases, must also be
a main cause for no increasing the FasL expression in
rejection condition. Further, studies will be needed to
elucidate the role of sCD95L in chronic rejection.
T lymphocytes can be separated into two sub-sets based
on their expression of the CD4 and CD8 molecules on the
cell surface. There are contradictory opinions on the relative
contribution of CD4+ and CD8+ T cells to allograft rejection.
Some animal models indicate that there is an absolute
requirement for CD4+ T cells in allogeneic rejection, whereas
in others CD4-depleted mice reject certain types of
allografts[32]. Haskova et al. used CD4- or CD8-deficient
knockout mice to investigate the role of T cell subsets in
allograft rejection. The results showed that CD4+ T cells
play a critical role in the rejection of corneal allografts,
whereas CD8+ T cells appear to be involved in the rejection
of skin allografts[33]. In our study, the CD4/CD8 ratio had
showed no significant difference among AR group, stable
group and healthy group. This finding is not compatible
with that of Sadeghi et al.[34] who showed that T lymphocyte
sub-populations were lower in rejecting than in non-rejecting
patients after renal transplantation. This difference may
result from different organ allograft. Further studies will
be needed to better understand the role of CD4 and CD8
T lymphocyte in the allograft rejection.
Human natural killer (NK) cells are defined by their
ability to lyse target cells without prior sensitization and
without restriction by major histocompatibility (MHC)
antigens. It has been shown that these cells play an important
role in immune defenses, especially after hematopoietic
transplantation[35]. In our study, we demonstrated that the
percentage of CD16+CD56+ cells did not change in AR
patients, implying that NK cells may not play an important
role in AR in liver transplantation or the immunosuppressive
therapy interfere with the generation of NK cells.
In conclusion, monitoring of CD95 on CD3+ T cells
and in plasma may provide an important clue to a better
understanding of the pathogenesis of liver graft rejection
and would be a helpful tool to develop new therapeutic
approaches for the prevention of AR by controlling the
CD95/CD95L signaling.
ACKNOWLEDGEMENTS ACKNOWLEDGEMENTS
ACKNOWLEDGEMENTSACKNOWLEDGEMENTS
ACKNOWLEDGEMENTS
We thank Dr. Wei Gao from Organ Transplantation Center
of Tianjin First Central Hospital for their clinical guidance.
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1704 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol March 21, 2005 Volume 11 Number 11