Changes of inducible protein-10 and regulated upon activation, normal T cell expressed and secreted protein in acute rejection of pancreas transplantation in rats.
ABSTRACT To investigate the role of IFN-gamma inducible protein -10 (IP-10) and regulated upon activation, normal T cell expressed and secreted (RANTES) protein in acute pancreatic allograft rejection in rats.
An experimental pancreas transplantation model was established using diabetic SD rats as the recipient, induced by applying streptozocin (STZ). Pancreas transplantation was performed with a physiologic method of portal venous and enteric drainage. Rats were divided into two groups, isograft group (group A, n = 24) and allograft group (group B, n = 24) in which either healthy SD rats or Wistar rats served as donors, respectively. Twelve diabetic or healthy SD rats were used as controls. At d 1, 4, 7, and 10 post transplantation, serum IP-10 and RANTES were assessed by ELISA and their expression in the allografts was determined by immunohistochemistry.
In group B (allograft group), the development of acute rejection was significantly correlated with increased serum concentration and tissue expression of IP-10 and RANTES, with a peak level at d 7 post transplantation. In contrast, there was no obvious change before and after transplantation in group A (isograft group).
Our study suggests a possible role of IP-10 and RANTES in acute rejection and early monitoring of chemokines may be helpful in predicting the outcome of pancreas transplantation.
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ABSTRACT: Cardiopulmonary bypass (CPB) leads to systemic and cardiac inflammation. Although the intraoperative blood measurement of some inflammatory cytokines has been recognized as an useful tool in clinical setting, postoperative management still represents a major problem; hence, knowledge about additional possible mediator(s) in time of this process would potentially turn in a clinical benefit. CXCL10 has been shown to be involved in cardiac immune-inflammatory processes. Herein, we aimed to investigate whether and how this chemokine could be a possible early mediator and indicator of inflammation development during CPB. Since cardiac, endothelial and immune cells are all sources of CXCL10, our purpose was also to investigate in vitro CXCL10 secretion pattern in time by those cell types. In thirteen patients undergoing CPB, CXCL10, CXCL9, IL-6, IL-8 and IL-10 have been measured by Elisa in serum withdrawn at eight different perioperative times. Sera from healthy subjects have been tested for comparison. The same cytokines have been measured by Elisa in time-course experiments onto human isolated cardiomyocytes, endothelial and CD4+T cells under inflammatory stimuli. TaqMan Real-Time RT-PCR and immunocytochemistry were used for endothelial cell characterization. CXCL10 level was higher in CPB patients before surgery as compared to healthy subjects; CXCL10 level raises earliest in serum of CPB patients and in isolated cardiomyocytes under inflammatory stimuli as compared to other cytokines. CXCL10 might represent a critical early factor in mediating systemic and local cardiac inflammatory response in subjects undergoing CPB, offering opportunity for future monitoring or therapeutic interventions.Biomedicine & Aging Pathology. 10/2012; 2(4).
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ABSTRACT: Progressive obliteration of the retinal microvessels is a characteristic of diabetic retinopathy. The resultant retinal ischemia leads to sight-threatening neovascularization and macular edema. Bone marrow-derived endothelial progenitor cells play a critical role in vascular maintenance and repair and forms the basis of cellular therapy for revascularization of ischemic myocardium and ischemic limbs. Emerging studies show potential of these cells in revascularization of ischemic retina and this review summarizes this possibility. We also report current pharmacological options to correct diabetes-associated defects in endothelial progenitor cells for their therapeutic transfer.Expert Review of Endocrinology & Metabolism 12/2009; 5(1):29-37.
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ABSTRACT: Rejection and ischemia are serious complications after liver transplantation. Early detection is mandatory, but specific markers are largely missing, particularly for rejection. The objective of this study was to explore the ability of microdialysis catheters inserted in liver grafts to detect and discriminate rejection and ischemia through postoperative measurements of inflammatory mediators. Microdialysis catheters with a 100-kDa pore size were inserted into 73 transplants after reperfusion. After the study's completion, complement activation product 5a (C5a), C-X-C motif chemokine 8 (CXCL8), CXCL10, interleukin-1 (IL-1) receptor antagonist, IL-6, IL-10, and macrophage inflammatory protein 1β were analyzed en bloc in all grafts with biopsy-confirmed rejection (n = 12), in grafts with vascular occlusion/ischemia (n = 4), and in reference grafts with a normal postoperative course of circulating transaminase and bilirubin levels (n = 17). The inflammatory mediators were elevated immediately after graft reperfusion and decreased toward low, stable values during the first 24 hours in nonischemic grafts. In grafts suffering from rejection, CXCL10 increased significantly (P = 0.008 versus the reference group and P = 0.002 versus the ischemia group) 2 to 5 days before increases in circulating alanine aminotransferase and bilirubin levels. The area under the receiver operating characteristic curve was 0.81. Grafts with ischemia displayed increased levels of C5a (P = 0.002 versus the reference group and P = 0.008 versus the rejection group). The area under the curve was 0.99. IL-6 and CXCL8 increased with both ischemia and rejection. In conclusion, CXCL10 and C5a were found to be selective markers for rejection and ischemia, respectively. Liver Transpl, 2012. © 2012 AASLD.Liver Transplantation 07/2012; · 3.79 Impact Factor
Jun Zhu, Ze-Kuan Xu, Yi Miao, Xun-Liang Liu, Hong Zhang
© 2006 The WJG Press. All rights reserved.
Key words: Pancreas transplantation; Chemokine; Rats
Zhu J, Xu ZK, Miao Y, Liu XL, Zhang H. Changes of inducible
protein-10 and regulated upon activation, normal T cell
expressed and secreted protein in acute rejection of
pancreas transplantation in rats. World J Gastroenterol
2006; 12(26): 4156-4160
In the past several decades, great success has been made
in the transplantation fi eld. However, the acute rejection
is still a tremendous obstacle to the development of the
transplantation. Acute allograft rejection is a complex
process comprising interrelated series of events, such as
the recognition of the allograft antigen, the activation
and proliferation of the leukocytes, the migration of the
leukocytes to the allograft. In the pathogenic process,
several kinds of inflammatory molecules are required,
such as proinfl ammatory cytokines, adherence molecules
and chemokines. The chemokines are a large family of
“chemoattractant cytokines”, including IFN-γ inducible
protein-10 (IP-10), regulated upon activation, normal T
cell expressed and secreted (RANTES), Mig, etc. They
play a critical role in directing leukocytes to the allo-
graft and in amplifying intragraft inflammation during
rejection[1,2]. In the past years it has become evident that
individual proinflammatory chemokines (such as IP-10,
RANTES) are indispensable in the rejection process
of heart transplantation, kidney transplantation, and
lung transplantation, compared to other chemokines[3-5].
However the roles of chemokines in pancreas
transplantation are still not well known and pancreas
transplantation has its own speciality that differs from
other organ transplantations. For example, it is well known
that pancreatic grafts are very susceptible to allograft
rejection because of the strong immunogenicity of
pancreas itself. To assess the changes of chemokines
IP-10 and RANTES in the acute rejection of pancreas
transplantation, we established the pancreas transplantation
model in rats fi rstly, using a physiologic method of portal
Changes of inducible protein-10 and regulated upon
activation, normal T cell expressed and secreted protein in
acute rejection of pancreas transplantation in rats
Jun Zhu, Ze-Kuan Xu, Yi Miao, Xun-Liang Liu, Department of
General Surgery, the First Affi liated Hospital of Nanjing Medical
University, Nanjing 210029, Jiangsu Province, China
Jun Zhu, Hong Zhang, Department of Experimental Surgery,
Xuzhou Medical College, Xuzhou 221004, Jiangsu Province,
Supported by a grant from the “135” Foundation of Jiangsu
Province, No. 2003-19
Correspondence to: Dr. Ze-Kuan Xu, Department of General
Surgery, The First Affiliated Hospital of Nanjing Medical
University, Nanjing 210029, Jiangsu Province,
Received: 2006-04-06 Accepted: 2006-04-24
AIM: To investigate the role of IFN-γ inducible protein
-10 (IP-10) and regulated upon activation, normal T
cell expressed and secreted (RANTES) protein in acute
pancreatic allograft rejection in rats.
METHODS: An experimental pancreas transplantation
model was established using diabetic SD rats as the
recipient, induced by applying streptozocin (STZ).
Pancreas transplantation was performed with a
physiologic method of portal venous and enteric
drainage. Rats were divided into two groups, isograft
group (group A, n = 24) and allograft group (group
B, n = 24) in which either healthy SD rats or Wistar
rats served as donors, respectively. Twelve diabetic or
healthy SD rats were used as controls. At d 1, 4, 7, and
10 post transplantation, serum IP-10 and RANTES were
assessed by ELISA and their expression in the allografts
was determined by immunohistochemistry.
RESULTS: In group B (allograft group), the development
of acute rejection was significantly correlated with
increased serum concentration and tissue expression
of IP-10 and RANTES, with a peak level at d 7 post
transplantation. In contrast, there was no obvious
change before and after transplantation in group A
CONCLUSION: Our study suggests a possible role
of IP-10 and RANTES in acute rejection and early
monitoring of chemokines may be helpful in predicting
the outcome of pancreas transplantation.
PO Box 2345, Beijing 100023, China World J Gastroenterol 2006 July 14; 12(26): 4156-4160
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Zhu J et al. Chemokines in pancreas transplantation 4157
venous and enteric drainage, and then evaluated the
severity of rejection, detected the concentration of serum
RANTES and IP-10 using ELISA kits, and examined the
expressive position and intensity of IP-10 and RANTES
in the allograft pancreas.
MATERIALS AND METHODS
The diabetic rats were induced fi rstly by single intravenous
injection of streptozocin (STZ; Sigma, USA) at a dose of
50 mg/kg of body weight, when the closed flock male
SD rats weighing 250-280 g were chosen (offered by the
Experimental Animal Center of Jiangsu Province, China).
After the STZ injection, the nonfasting blood glucose
and urine glucose of the rats were assayed before the
inducement and measured on alternate day. Only rats with
nonfasting blood glucose exceeding 16.8 mmol/L and the
strong positive reaction of the urine glucose were selected
as recipients. The closed flock male Wistar rats (offered
by the Silaike Co. Ltd, Shanghai, China) and healthy SD
rats weighing 220-250 g served as donors. The donors
and recipients were matched under the condition that the
weights of the recipients were more than that of donors
by about 30 g.
Grouping of experimental rats
Rats were divided into two groups, isograft group (group
A, n = 24) and allograft group (group B, n = 24), in which
either healthy SD rats or Wistar rats served as donors,
respectively. Twelve diabetic or healthy SD rats were used
Surgical procedure and collection of specimen
A physiologic method for pancreas transplantation was
adopted, in which the vein was reconstructed by end-
to-side anastomosis between the donor portal vein
and the recipient superior mesenteric vein, and arterial
reconstruction was carried out by end-to-side anastomosis
of the donor to the recipient abdominal aorta, and enteric
drainage was performed by a side-to-side anastomosis
between the duodenum of donors and that of recipients.
The level of the recipient’s blood glucose below 11.2
mmol/L at 1 d post operation was regarded as successful
transplantation. The recipients were sacrifi ced at 1, 4, 7,
10 d (n = 6 animals/time point) after transplantation. The
12 rats in control groups were killed at the beginning of
the experiment. Blood samples were collected and placed
quietly for clotting for 2 h at room temperature before
centrifuging for 30 min at 1000 × g, then the serum
was pipetted immediately and stored at -70℃. After
representative portions of pancreas grafts were removed,
some of them were immediately snap-frozen in liquid
nitrogen for immunohistology and the rest were fi xed in
10% formalin for histopathological examination.
The samples of pancreas grafts were fixed, dehydrated,
embedded, sliced, and stained with hematoxylin and
eosin following the routine proposal. The classification
of acute rejection was stated according to the Nakhleh
Classifi cation Criterion.
Determination of serum IP-10 and RANTES
ELISA kits (TPI INC., USA) were used for the
determination of serum IP-10 and RANTES, and
the procedure was strictly according to the protocol
recommended by the manufacturers. The results were
expressed as the quantity per mL serum.
For immunohistology, 10 μm frozen sections of pancreas
were prepared, fi xed in acetone for 10 min, dried in the
airy place, and incubated with goat polyclonal IP-10
antibodies and rabbit polyclonal RANTES antibodies
respectively. Then, the sections were incubated with rabbit
anti-goat IgG and goat anti-rabbit IgG respectively. All the
reagents were offered by Santa Cruz Co, USA. The cells
stained clearly were regarded as positive ones. According
to the percentage of positive cells in the whole infi ltrating
immune cells, the results of immunohistology were
expressed in four grades: negative (the rate of positive
cells < 5%), mild positive (the rate of positive cells > 5%
and < 25%), moderate positive (the rate of positive cells >
25% and < 50%), strong positive (the rate of positive cells
The concentration of serum IP-10 and RANTES were
expressed as mean ± SD. The signifi cance of differences
was tested using either t-test for means of two samples
or analysis of Variance and q-test for means of multiple
samples. P < 0.05 was considered as signifi cant.
Classifi cation of acute rejection
The acute rejection was classifi ed according to the criterion
stated by Nakhleh. In this study, a mild edema appeared
around the islet and the acinus 1 d post transplantation
both in the allograft and isograft groups. The edema
disappeared and no evident rejection was found at 4, 7,
and 10 d after the operations in the isograft group, though
evident rejection appeared in the allograft group (Table 1).
Changes of serum IP-10 and RANTES
In the allograft group, serum IP-10 concentration was
elevated signifi cantly since 4 d and peaked at 7 d after the
Table 1 Classifi cation of acute rejection in allograft group post
Nakhleh Classifi cation Criterion, Grade Ⅰ: no rejection present, Grade Ⅱ:
mild lymphoplasmacellular infi ltration or endothelialitis, Grade Ⅲ: moderate
lymphoplasmacellular infiltration or arteritis/vasculitis, Grade Ⅳ: severe
lymphoplasmacellular infi ltration or fi brinoid necrosis.
operations, compared to the control group (P < 0.05).
However, no significant difference was found between
the isograft group and the control group at the four
corresponding phases. The tendency of serum RANTES
was similar to that of IP-10, only showing a sharp increase
at 1 d after the transplantation in the isograft group (P <
0.05) compared with the control group (Tables 2 and 3).
Expression of IP-10 and RANTES in the pancreas grafts
There were no detectable expressions of IP-10 and
RANTES protein in the normal pancreas. Mild expression
was observed at 1 d after the operation both in the
allografts and isografts (Figures 1 and 2). At 4 d after
the transplantation, expression of IP-10 and RANTES
appeared moderately in the allografts (Figures 3 and 4),
whereas their expression could not be detected in the
isografts. At 7 d after the operation, the positive expression
was strongest in the allografts (Figures 5 and 6). Besides,
they were expressed in different places. IP-10 was gathered
around the vessels; RANTES was clearly expressed in
the lymphocytes in interstitial tissues and concentrated in
the narrow space between the infl ammatory and normal
Acute rejection is a very complicated process, in which the
cellular and humoral immune mechanisms are involved.
In the process of acute rejection, the cellular immune
reaction is of primary importance. The circulating
lymphocytes firstly adhere to the vessel endothelium,
then penetrate the vessel wall, cross the interstitial tissue,
migrate into the graft, and finally destroy the graft.
In these processes, the chemokines are indispensable.
Table 2 Concentration of serum IP-10 in isograft, allograft and
control groups (ng/L)
aP < 0.05 vs control group.
1 d4 d 7 d 10 d
Allograft group 43.34 ± 15.29 66.26 ± 11.08ａ83.28 ± 16.44ａ70.08 ± 17.65ａ
Control group28.76 ± 7.41
40.88 ± 10.76 36.74 ± 10.3338.13 ± 12.7331.83 ± 7.55
Table 3 Concentration of serum RANTES in isograft, allograft
and control groups (ng/L)
aP < 0.05 vs control group.
Group 1 d 4 d 7 d 10 d
Allograft 388.48 ± 104.45a586.72 ± 90.06a746.28 ± 92.64a631.49 ± 106.34a
Control 251.18 ± 44.94
357.87 ± 63.26a274.77 ± 58.22265.87 ± 43.40267.55 ± 48.91
Figure 1 IP-10 immunohistochemical staining of pancreas allografts. Mild
expression was observed at 1 d post transplantation (× 200).
Figure 2 RANTES immunohistochemical staining of pancreas allografts. Mild
expression was observed at 1 d post transplantation (× 200).
Figure 3 IP-10 immunohistochemical staining of pancreas allografts. Moderate
expression was observed at 4 d post transplantation (× 200).
Figure 4 RANTES immunohistochemical staining of pancreas allografts.
Moderate expression was observed at 4 d post transplantation (× 200).
4158 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol July 14, 2006 Volume 12 Number 26
Chemokines accelerate the migration and activation of
the leucocytes and lymphocytes through the interaction
between the chemokines and their receptors, and play a
key role in recruitment of infl ammatory cells into an organ
Chemokines are a group of 8-11 kDa proteins, owning
four conservative tyrosines in their primary sequence.
According to the positional relation between the first
tyrosine and the second one, the family are divided into
four subfamilies, namely, CXC subfamily, CC subfamily, C
subfamily and CX3C subfamily.
IP-10 is one of CXC subfamily, mainly coming from
activated fibroblasts, monocytes, endothelial cells and T
cells. By the interaction between IP-10 and its receptor
(CXCR3), IP-10 function in the recruitment of activated
Th1 cells specifi cally and amplify the Th1 reaction. It has
been reported that IP-10 and CXCR3 were detected in
the allograft in the lung and heart transplantation, and
the quantities of IP-10 and CXCR3 are positively related
with the severity of rejection response. The latest
study performed by Kanmaz et al showed an important
correlation between urinary excretion of IP-10 and acute
rejection in baboon kidney transplantation and indicated
that urinary IP-10 might be a more accurate predictive
parameter than serum creatinine to monitor the occurrence
of acute rejection of renal transplant.
RANTES belongs to the CC subfamily, produced
and secreted by the CD8+ T cells, endothelial cells, and
fi broblasts, directing the recruitment of T cells, monocytes,
eosinophils to the local tissue. It has been confirmed
that RANTES is closely related to the pathogenesis
of rejection in heart, lung, kidney transplantation, etc.
Mulligan et al found that the protein of RANTES was
detectable 6-8 d after the heart transplantation in mice in
heterogeneous group. However, the results were opposite
in the homogeneous heart transplantation. Sekine et al
reported that the mRNA of RANTES was closely
positively correlated with the number of infiltrating
monocytes, using the models of lung transplantation.
Similar results were also obtained in liver transplantation.
Some experiments have succeeded in prolonging the
functional time of the allografts by inhibiting the
interaction between the chemokines and their receptors.
Hancock et al reported that the survival phase was elon-
gated, provided that the IP-10 knockout mice served as
the donors. Yun et al demonstrated that the use of Met-
RANTES, the specific antagonist to RANTES, could
alleviate the infi ltration of CD4+ and CD8+ lymphocytes
and attenuate allograft rejection in mice. It seems that
IP-10 and RANTES play a pivotal role in the rejection of
In our experiments, there was no detection of IP-10
and RANTES protein in the normal pancreas. In the
isograft group, the proteins of IP-10 and RANTES were
detected weakly at 1 d, and restored to the normal level
since 4 d post transplantation. In the allograft group,
the expression of IP-10 and RANTES protein were also
upregulated in the primary phase of acute rejection, and
then developed with the severity of the rejection reaction.
The trend of serum IP-10 and RANTES was the same as
that in the graft. It seems that the detection of IP-10 and
RANTES benefi ts the early diagnosis of acute rejection.
We also noticed that the expressing loci and pattern
of IP-10 and RANTES was obviously different. IP-10
was gathered around the vessels; meanwhile, RANTES
was clearly expressed in the lymphocytes in the interstitial
tissues and crowded in the narrow space between the
inflammatory and normal tissues. This result perhaps
demonstrated that IP-10 and RANTES play different roles
in the acute rejection. IP-10 is a key molecule in directing
the lymphocytes to penetrate the endothelium and migrate
across the tissue. RANTES, produced by the cells in the
infl ammatory tissues, can be recognized and bound with
its receptors on the surface of passage macrophage, T
cells. The activated T cells unregulated the expression of
RANTES, and more infl ammatory cells were attracted into
the graft. The process was cycled and amplifi ed and the
infl ammation aggravated.
In summary, IP-10 and RANTES play a critical role in
the acute rejection after pancreas transplantation. Further
research will help the understanding of the pathogenesis
of acute rejection. IP-10 and RANTES will be a tool for
the early diagnosis and a target for the therapy of acute
Melter M, Exeni A, Reinders ME, Fang JC, McMahon G, Ganz
P, Hancock WW, Briscoe DM. Expression of the chemokine
Figure 6 RANTES immunohistochemical staining of pancreas allografts. Strong
positive expression was observed at 7 d post transplantation (× 200).
Figure 5 IP-10 immunohistochemical staining of pancreas allografts. Strong
positive expression was observed at 7 d post transplantation (× 200).
Zhu J et al. Chemokines in pancreas transplantation 4159
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S- Editor Wang J L- Editor Zhu LH E- Editor Liu WF
4160 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol July 14, 2006 Volume 12 Number 26