Extract
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Introduction
CD4+ Th cells can differentiate into two subsets, Th1 and Th2 cells. Th1 cells produce cytokines such as interleukin
(IL)-2, interferon (IFN)-g, and tumor necrosis factor
(TNF)-a and often orchestrate the cellular immune
responses noted in certain organ specific autoimmune diseases, allograft rejection,
and delayed-type hypersensitivity responses. In contrast, Th2
cells secrete IL-4, IL-5, IL-6, and IL-10 cytokines and lead to the production of antibodies contributing to humoral immunity. The
balance between Th1 and Th2 cells determines an overcome of immune
response[1].
Previous studies have shown that allograft rejections including the heart, skin and islet are usually associated with a
Th1-type response. In contrast, the tolerant allograft in recipients often manifested a Th2-type
response[2_5]. When IFN-g, gene knockout (KO) mice were used as graft recipients, the allograft survival was found to be highly conditional and both the host
strain and experimental conditions could influence the
results[6]. When IL-4-deficient mice were used as an allograft recipient
accompanied by CTLA4/Fc treatment, the allograft rejection was inhibited as compared to the control, wild-type (WT)
mice[7].
IL-12 and IL-23 are believed to be Th1 initiators playing an essential role in Th1
development[8_10]. The cytokines are composed of a p35 and p40 subunit for IL-12 and p19 and p40 subunit for IL-23. They are produced primarily by activated
antigen-presenting cells, such as dendritic cells and macrophages, proinflammatory natural-killer (NK) and activated T cells,
and induced cell-mediated
immunity[11]. The p40 subunit KO mice results in a lack of IL-12 and IL-23 and consequently leads
to a defect in inducing Th1 cell response in autoimmune diseases, such as experimental autoimmune encephalomyelitis
(EAE)and experimental autoimmune uveoretinitis
(EAU)[12,13]. The data demonstrate that in IL-12p40 transgenic mice the
development of diabetes was exacerbated in non-obese diabetic (NOD)
mice[14]. The heart allograft rejection was accelerated when
recipient mice were injected with dendritic cell (DC) expressing
IL-12p40[15]. It is unclear whether islet allograft survival is
postponed in IL-12p40-deficient mice.
In this study, we investigated whether IL-12p40-deficient mice prevented islet allograft rejection in the streptozotocin
(STZ)-induced diabetes mouse model. Our data showed that in an STZ-induced type-1 diabetes model, the survival time of
islet allograft was similar in IL-12p40-deficient mice and WT mice.
Materials and methods
Mice Six-to eight-week-old female C57BL/6
(H2b), BALB/c
(H2d), IL-12p40-deficient
(P40KO,C57B6.129S1-Il12btm1Jm/J) mice were
purchased from the Jackson Laboratory (Bar Harbor, ME, USA). Animals were
kept in a specific pathogen-free facility at the Chinese
Academy of Sciences. Animal care and use were in compliance with
institutional guidelines.
STZ treatment and diabetes induction Recipient mice
were given diabetes by a single ip injection of STZ (150
mg/kg for C57 and 200 mg/kg for IL-12p40 deficient mice ). STZ (Fluka, St Louis, MO) was dissolved in sodium citrate buffer (pH 4.5).
Diabetes was defined as a plasma concentration of glucose >16.7 mmol/L within 2 d after the end of STZ
treatment[16]. Plasma glucose concentration was measured using a Medisense Optium blood detector (Abbott Laboratories,MediSense products,
Bedfold, USA).
Islet isolation and transplantation Islets were isolated from female BALB/c
(H-2d) donors by using a filtered method as
described in a previous study[17]. The pancreas was digested by collagenase P (Roche Diagnostics Corporation Indianapolis,
IN, USA) for 12 min, and followed by filtering with a 300 µm strainer. The filtered part was poured through a 100 µm cell
strainer (BD Falcon, Bedfold, USA) to enrich the islets. Finally,
500 islets (diameter varied between 100 µm and 300 µm)
were handpicked and transplanted into the renal subcapsular space of STZ-induced diabetic
recipients[18].
A graft transplantation was considered successful when
the plasma glucose concentration was reduced to 16.7
mmol/L within 48 h. If not, the mouse was considered to be a technical failure and was excluded from analysis. Graft rejection was
defined as the recurrence of a plasma glucose concentration >16.7 mmol/L on 2 successive
days. Islet allograft function was confirmed by unilateral nephrectomy of the kidney bearing the transplant and documentation of the reappearance of diabetes.
ELISA The sample of mouse serum was collected and frozen at -70 °C until it was detected. Briefly, 20 µg/mL of EQ31-BSA
(the C-peptide conjugated with BSA carrier) was coated with 100 µL/well in a 96-well Nunc Immunoplate (Nunc, Roskilde,
Denmark) overnight at 4 °C. The plates were blocked with blocking buffer (PBS with 3% gelatin) for 2 h at 37 °C. Sample 100
µL (containing 90 µL serum and 10 µL C-peptide antibody) was added and incubated at 37 °C for 2 h. Then a second
antibody horseradish peroxidase (HRP)-conjugated goat anti-mouse immunoglobulin (Ig)-G (BD
PharMingen, San Diego, CA, USA) at 100 µL/well was added and
OD values were measured with a microplate autoreader (Biotek, Vermont,USA) at 450
nm. The standard curve for C peptide was created with different concentrations of C peptide and was used to measure C peptide
concentrations in tested sera samples by competitive ELISA.
Immunohistopathology Islet graft was embedded in
optimal cutting temperature compound (OCT) and frozen at -20
°C. The sections of 10-µm thickness were kept at -70 °C until
staining. CD4+ and CD8+ cells were determined by immunohistochemistry
with the specific antibodies (BD PharMingen).
Real-time PCR Total RNA was isolated from graft. An mRNA expression of
IFN-g and IL-4 was determined by real-time PCR using SYBR
Green Master Mix (Applied Biosystems, Foster City, USA) as described in a previous
study[19]
Statistical analysis The log-rank test of nonparametric analysis (Instat software; Graph Pad, San Diego, CA, USA)was
used to analyze graft survival data. The differences in graft survival were analyzed by a Kaplan-Meier test. The experiments
were usually repeated three times.
Results
Establishment of islet allograft model Initially,
C57BL/6 and IL-12p40-deficient mice served as recipient mice and STZ was
used to induce diabetes in the mice. We found that the different genetic background had a great impact on
diabetes induction, which was consistent with otherĄ¯s
reports[20]. Based on our primary experiments, the optimal dose of STZ to induce diabetes
in C57BL/6 mice is 150 mg/kg, but it was 200 mg/kg in IL-12p40-deficient mice.
After STZ ip injection, the glucose in the blood increased rapidly and remained at a relative high level. However, it
remained normal in the vehicle control group. Based on this protocol, the diabetes was developed within a few days in
STZ-treated mice. If the sick mice were not treated with islet transplantation, the mice began to die on d 10 after STZ treatment
(Figure 1A).
Five hundred islets freshly isolated from BALB/c or C57BL/6 mice were transplanted into the renal subcapsular space of
C57BL/6 mice. Then the glucose in the blood was detected. The data showed that, after transplantation of the islets, the
enhanced glucose in the recipient mice dropped to the normal level within 2 d (Figure 1B). It was obvious that the glucose
concentration was increased and exceeded 16.7 mmol/L on d 10 following the islet transplantation and remained
hyperglycemic when the islet allograft from Balb/c mice (allogeneic group) was rejected completely. But the islets from C57BL/6 mice
(syngeneic group) functioned well and the blood glucose remained normal until 20 d after islet transplantation. In contrast,
when HanksĄ¯ solution served as a control (vehicle group) in the transplantation, the hyperglycemia was not changed.
To confirm the islet rejection, the left kidneys from islet transplanted mice were removed on d 5 (non-rejected islet) and d
12 following the transplantation. The tissue histopathology was stained with HE. Only intact islets could be seen in the
tissues of non-rejected islets, but could not be seen in rejected islet (data not shown).
It was reported that the concentration of C-peptide in the blood was reduced when the diabetes
occurred[21]. The concentration of C-peptide was detected in the mice serum from different types of the disease. The C-peptide concentration
was consistent with diabetes. In control C57BL/6 mice, the C-peptide secretion was normal. After diabetes was induced, the
C-peptide concentration was decreased. When islet transplantation was successful, the secretion of the C-peptide was
rescued and then it returned to low levels after the allograft was rejected completely. The data indicates that C-peptide
concentration is a good marker to reflect the stature of insulin secretion
in vivo (Figure 1C).
Role of IL-12p40 gene in islets allograft transplantation
Islets were isolated from major histocompatibility complex
(MHC)-mismatched BALB/c mice and then they were transplanted into IL-12p40- deficient or wild-type C57BL/6 mice. As
shown in Figure 2A and 2B, the rejection rate and survival mean days for islet transplantation were similar
between WT and IL-12p40-deficient mice. In the control group,
after islet transplantation, the mean survival time was
9.7±2.1 d (n=6). Similarly, it was 10.0±2.9 d
(n=7) in the IL-12p40-deficient mice. The results suggested that after islet transplantation,
the C57BL/6 WT and IL-12p40-deficient mice rejected the allograft at a similar rate, which was not as anticipated.
Immunohistopathology Previous studies have revealed that T cells play an important role during the islet allograft
rejection and that the deficiency of IL-12p40 can affect the function of
CD4+ and CD8+ T cells. In this study, we observed the
infiltration of CD4+ and
CD8+ T cells in the allograft. On d 9 after transplantation, the infiltration of
CD4+ and CD8+ cells in the grafts was similar in the recipients of WT and IL-12p40-deficient mice (Figure
3).
IL-4 and IFN-g expression in the graft As Th1 and Th2 cells are believed to play a crucial role in determining the islet
rejection, IL-4 and IFN-g expressions in infiltrating T cells in the grafts were measured by real-time PCR. As we expected,
IL-4 and IFN-g expressions were similar in the grafts from WT and IL-12p40-deficient mice (Figure 4).
Discussion
Since the discovery of the cross-regulating Th1 and Th2 phenotypes, there has been great anticipation that a Th1 to Th2
immune deviation may be critical in the acquisition of transplantation tolerance. This is because IL-12 is a major
inducer and a pivotal regulator in the generation of Th1
cells[22]. It is also believed that IL-12 plays a key role in the destruction of
insulin-producing cells during the development of autoimmune diabetes
with a higher production of IFN-g and lower production of
IL-4, which is representative of a Th1-type
response[23_25]. We are interested in studying the roles of IL-12 and IL-23 in
allograft rejection, which is regarded as a Th1 cell-mediated
response[26,27]. Allograft rejection in unmodified recipients is
often associated with a Th1-type response. Moreover, allograft recipients that are treated with tolerant immunosuppressive
regimens often manifest a Th2-type response during the treatment
period[3_5].
In the present study, we addressed whether Th1 to Th2 immune deviation in IL-12p40-deficient mice could eliminate the
severity of allograft rejection based on the fact that Th1 cells are essential in induced autoimmune
diseases[28] and the observation in
CCR5_/_ mice of an obvious prolongation of islet allograft survival and a switch to Th2
response[18]. We predicted that IL-12p40 might play a determining role in islet allograft rejection in a STZ-induced diabetic mouse model.
Surprisingly, islet allograft from BALB/c mice survived well in IL-12p40- deficient mice (C57BL/6 background) as compared to
wild-type control mice, their mean survival time was similar and the values were 10.0±2.9 d and 9.7±2.1 d, respectively. In
addition, there was no significant difference in the infiltration of CD4+ and CD8+ T cell and the cytokines expression
(IFN-g and IL-4) in transplanted grafts.
According to our observations, although tolerance therapy in some animal models often skewed the immune
activation toward a Th2-dominated response[
26,27], a Th1 to Th2 immune deviation does not uniformly permit the acquisition
of transplant tolerance. This new finding leads us to make an explanation about the mechanisms of allograft
rejection[29,30]. First, we have noticed that the induction of permanent engraftment or a state of allograft tolerance through the administration
of long-acting Th2 cytokines (ie, IL-4-Ig and IL-10-Ig fusion proteins) or immune deviation via the application of IL-12
antagonists have failed in the heart
allograft[30,31]. It is obvious that the strategies, which are extremely effective in dampening
autoimmunity, have not been proven to be effective in
transplantation[32_36]. In accordance with our results, although
anti-IL-12 was used to deviate Th1 to Th2, anti-IL-12 treatment was totally ineffective in prolonging the engraftment of
MHC-mismatched islet allografts. While the impact of anti-IL-12 upon the pattern of cytokine expression was consistent, the
differences were noted in the impact of such therapy upon the duration of engraftment in MHC-mismatched versus
MHC-matched conditions. It is concluded that regarding to the model of Ag presentation and the responding T cell clone number,
the allograft response to MHC-matched rather than minor Ag-mismatched allografts may more closely resemble the response
to auto-antigens[37]. It should be pointed out that a manifestation of a Th2-type response is not equal to an induced Th1 to
Th2 immune deviation manipulation. If they have equal effects
in vivo, logically, the Th2 biased polarization is only to induce
weak tolerance rather than a sufficient condition to block immune response.
The evidence indicates that Th2 cytokine production to either graft survival or rejection is based on assessing intragraft
cytokine gene expression by RT-PCR or identifying graft infiltrating cells that stain for cytokine protein by
immunohistochemistry within allografts. As mentioned by Piccotti
et al[29], these assays do not take into account the antigen specificity
of the cells producing cytokines. Limiting dilution analysis studies have revealed that the frequency of donor-specific T cells
infiltrating grafts is very low. Hence, RT-PCR may detect cytokine mRNA produced by irrelevant cells that are trafficking
through the graft. We also found that the cytokines such as
IFN-g and IL-4 expressed by the grafts had no difference. This
observation suggested that the evaluation of intragraft cytokine profiles for Th1/Th2 dominance should be viewed carefully.
We accepted that all of the cytokine profile reflected a Th2 bias in the graft location, there was no solid evidence to indicate
that Th2 cells are really protective as we had previously thought.
It should be pointed out that IL-12p40 is the common-chain of IL-12 and IL-23. Thus, the IL-12p40-deficient mice have
both cytokines knocked out. Recently, the important role of IL-23 in autoimmune diseases and pathological inflammatory
processes has been unraveled. IL-23 drives the development of a novel T-cell subset characterized by the production of
IL-17 (ThIL-17), which plays a central role in mediating chronic inflammatory
responses[38]. It is not clear whether IL-23 will have
any impact on allograft rejection. Our results suggested that a lack of IL-23 in p40-deficient mice did not inhibit islet
transplantation.
In conclusion, the islet allograft survival is not prolonged in IL-12p40-deficient mice as compared to WT controls. We
conclude that Th cell activation rather than Th1/Th2 polarization plays the major role in controlling allograft rejection. The
precise mechanism for the failure of Th1 to Th2 immune deviation in allograft rejection in MHC-mismatched allograft
recipients will require further investigation.
Introduction
CD4+ Th cells can differentiate into two subsets, Th1 and Th2 cells. Th1 cells produce cytokines such as interleukin
(IL)-2, interferon (IFN)-g, and tumor necrosis factor
(TNF)-a and often orchestrate the cellular immune
responses noted in certain organ specific autoimmune diseases, allograft rejection,
and delayed-type hypersensitivity responses. In contrast, Th2
cells secrete IL-4, IL-5, IL-6, and IL-10 cytokines and lead to the production of antibodies contributing to humoral immunity. The
balance between Th1 and Th2 cells determines an overcome of immune
response[1].
Previous studies have shown that allograft rejections including the heart, skin and islet are usually associated with a
Th1-type response. In contrast, the tolerant allograft in recipients often manifested a Th2-type
response[2_5]. When IFN-g, gene knockout (KO) mice were used as graft recipients, the allograft survival was found to be highly conditional and both the host
strain and experimental conditions could influence the
results[6]. When IL-4-deficient mice were used as an allograft recipient
accompanied by CTLA4/Fc treatment, the allograft rejection was inhibited as compared to the control, wild-type (WT)
mice[7].
IL-12 and IL-23 are believed to be Th1 initiators playing an essential role in Th1
development[8_10]. The cytokines are composed of a p35 and p40 subunit for IL-12 and p19 and p40 subunit for IL-23. They are produced primarily by activated
antigen-presenting cells, such as dendritic cells and macrophages, proinflammatory natural-killer (NK) and activated T cells,
and induced cell-mediated
immunity[11]. The p40 subunit KO mice results in a lack of IL-12 and IL-23 and consequently leads
to a defect in inducing Th1 cell response in autoimmune diseases, such as experimental autoimmune encephalomyelitis
(EAE)and experimental autoimmune uveoretinitis
(EAU)[12,13]. The data demonstrate that in IL-12p40 transgenic mice the
development of diabetes was exacerbated in non-obese diabetic (NOD)
mice[14]. The heart allograft rejection was accelerated when
recipient mice were injected with dendritic cell (DC) expressing
IL-12p40[15]. It is unclear whether islet allograft survival is
postponed in IL-12p40-deficient mice.
In this study, we investigated whether IL-12p40-deficient mice prevented islet allograft rejection in the streptozotocin
(STZ)-induced diabetes mouse model. Our data showed that in an STZ-induced type-1 diabetes model, the survival time of
islet allograft was similar in IL-12p40-deficient mice and WT mice.
Materials and methods
Mice Six-to eight-week-old female C57BL/6
(H2b), BALB/c
(H2d), IL-12p40-deficient
(P40KO,C57B6.129S1-Il12btm1Jm/J) mice were
purchased from the Jackson Laboratory (Bar Harbor, ME, USA). Animals were
kept in a specific pathogen-free facility at the Chinese
Academy of Sciences. Animal care and use were in compliance with
institutional guidelines.
STZ treatment and diabetes induction Recipient mice
were given diabetes by a single ip injection of STZ (150
mg/kg for C57 and 200 mg/kg for IL-12p40 deficient mice ). STZ (Fluka, St Louis, MO) was dissolved in sodium citrate buffer (pH 4.5).
Diabetes was defined as a plasma concentration of glucose >16.7 mmol/L within 2 d after the end of STZ
treatment[16]. Plasma glucose concentration was measured using a Medisense Optium blood detector (Abbott Laboratories,MediSense products,
Bedfold, USA).
Islet isolation and transplantation Islets were isolated from female BALB/c
(H-2d) donors by using a filtered method as
described in a previous study[17]. The pancreas was digested by collagenase P (Roche Diagnostics Corporation Indianapolis,
IN, USA) for 12 min, and followed by filtering with a 300 µm strainer. The filtered part was poured through a 100 µm cell
strainer (BD Falcon, Bedfold, USA) to enrich the islets. Finally,
500 islets (diameter varied between 100 µm and 300 µm)
were handpicked and transplanted into the renal subcapsular space of STZ-induced diabetic
recipients[18].
A graft transplantation was considered successful when
the plasma glucose concentration was reduced to 16.7
mmol/L within 48 h. If not, the mouse was considered to be a technical failure and was excluded from analysis. Graft rejection was
defined as the recurrence of a plasma glucose concentration >16.7 mmol/L on 2 successive
days. Islet allograft function was confirmed by unilateral nephrectomy of the kidney bearing the transplant and documentation of the reappearance of diabetes.
ELISA The sample of mouse serum was collected and frozen at -70 °C until it was detected. Briefly, 20 µg/mL of EQ31-BSA
(the C-peptide conjugated with BSA carrier) was coated with 100 µL/well in a 96-well Nunc Immunoplate (Nunc, Roskilde,
Denmark) overnight at 4 °C. The plates were blocked with blocking buffer (PBS with 3% gelatin) for 2 h at 37 °C. Sample 100
µL (containing 90 µL serum and 10 µL C-peptide antibody) was added and incubated at 37 °C for 2 h. Then a second
antibody horseradish peroxidase (HRP)-conjugated goat anti-mouse immunoglobulin (Ig)-G (BD
PharMingen, San Diego, CA, USA) at 100 µL/well was added and
OD values were measured with a microplate autoreader (Biotek, Vermont,USA) at 450
nm. The standard curve for C peptide was created with different concentrations of C peptide and was used to measure C peptide
concentrations in tested sera samples by competitive ELISA.
Immunohistopathology Islet graft was embedded in
optimal cutting temperature compound (OCT) and frozen at -20
°C. The sections of 10-µm thickness were kept at -70 °C until
staining. CD4+ and CD8+ cells were determined by immunohistochemistry
with the specific antibodies (BD PharMingen).
Real-time PCR Total RNA was isolated from graft. An mRNA expression of
IFN-g and IL-4 was determined by real-time PCR using SYBR
Green Master Mix (Applied Biosystems, Foster City, USA) as described in a previous
study[19]
Statistical analysis The log-rank test of nonparametric analysis (Instat software; Graph Pad, San Diego, CA, USA)was
used to analyze graft survival data. The differences in graft survival were analyzed by a Kaplan-Meier test. The experiments
were usually repeated three times.
Results
Establishment of islet allograft model Initially,
C57BL/6 and IL-12p40-deficient mice served as recipient mice and STZ was
used to induce diabetes in the mice. We found that the different genetic background had a great impact on
diabetes induction, which was consistent with other's
reports[20]. Based on our primary experiments, the optimal dose of STZ to induce diabetes
in C57BL/6 mice is 150 mg/kg, but it was 200 mg/kg in IL-12p40-deficient mice.
After STZ ip injection, the glucose in the blood increased rapidly and remained at a relative high level. However, it
remained normal in the vehicle control group. Based on this protocol, the diabetes was developed within a few days in
STZ-treated mice. If the sick mice were not treated with islet transplantation, the mice began to die on d 10 after STZ treatment
(Figure 1A).
Five hundred islets freshly isolated from BALB/c or C57BL/6 mice were transplanted into the renal subcapsular space of
C57BL/6 mice. Then the glucose in the blood was detected. The data showed that, after transplantation of the islets, the
enhanced glucose in the recipient mice dropped to the normal level within 2 d (Figure 1B). It was obvious that the glucose
concentration was increased and exceeded 16.7 mmol/L on d 10 following the islet transplantation and remained
hyperglycemic when the islet allograft from Balb/c mice (allogeneic group) was rejected completely. But the islets from C57BL/6 mice
(syngeneic group) functioned well and the blood glucose remained normal until 20 d after islet transplantation. In contrast,
when Hanks' solution served as a control (vehicle group) in the transplantation, the hyperglycemia was not changed.
To confirm the islet rejection, the left kidneys from islet transplanted mice were removed on d 5 (non-rejected islet) and d
12 following the transplantation. The tissue histopathology was stained with HE. Only intact islets could be seen in the
tissues of non-rejected islets, but could not be seen in rejected islet (data not shown).
It was reported that the concentration of C-peptide in the blood was reduced when the diabetes
occurred[21]. The concentration of C-peptide was detected in the mice serum from different types of the disease. The C-peptide concentration
was consistent with diabetes. In control C57BL/6 mice, the C-peptide secretion was normal. After diabetes was induced, the
C-peptide concentration was decreased. When islet transplantation was successful, the secretion of the C-peptide was
rescued and then it returned to low levels after the allograft was rejected completely. The data indicates that C-peptide
concentration is a good marker to reflect the stature of insulin secretion
in vivo (Figure 1C).
Role of IL-12p40 gene in islets allograft transplantation
Islets were isolated from major histocompatibility complex
(MHC)-mismatched BALB/c mice and then they were transplanted into IL-12p40- deficient or wild-type C57BL/6 mice. As
shown in Figure 2A and 2B, the rejection rate and survival mean days for islet transplantation were similar
between WT and IL-12p40-deficient mice. In the control group,
after islet transplantation, the mean survival time was
9.7±2.1 d (n=6). Similarly, it was 10.0±2.9 d
(n=7) in the IL-12p40-deficient mice. The results suggested that after islet transplantation,
the C57BL/6 WT and IL-12p40-deficient mice rejected the allograft at a similar rate, which was not as anticipated.
Immunohistopathology Previous studies have revealed that T cells play an important role during the islet allograft
rejection and that the deficiency of IL-12p40 can affect the function of
CD4+ and CD8+ T cells. In this study, we observed the
infiltration of CD4+ and
CD8+ T cells in the allograft. On d 9 after transplantation, the infiltration of
CD4+ and CD8+ cells in the grafts was similar in the recipients of WT and IL-12p40-deficient mice (Figure
3).
IL-4 and IFN-g expression in the graft As Th1 and Th2 cells are believed to play a crucial role in determining the islet
rejection, IL-4 and IFN-g expressions in infiltrating T cells in the grafts were measured by real-time PCR. As we expected,
IL-4 and IFN-g expressions were similar in the grafts from WT and IL-12p40-deficient mice (Figure 4).
Discussion
Since the discovery of the cross-regulating Th1 and Th2 phenotypes, there has been great anticipation that a Th1 to Th2
immune deviation may be critical in the acquisition of transplantation tolerance. This is because IL-12 is a major
inducer and a pivotal regulator in the generation of Th1
cells[22]. It is also believed that IL-12 plays a key role in the destruction of
insulin-producing cells during the development of autoimmune diabetes
with a higher production of IFN-g and lower production of
IL-4, which is representative of a Th1-type
response[23_25]. We are interested in studying the roles of IL-12 and IL-23 in
allograft rejection, which is regarded as a Th1 cell-mediated
response[26,27]. Allograft rejection in unmodified recipients is
often associated with a Th1-type response. Moreover, allograft recipients that are treated with tolerant immunosuppressive
regimens often manifest a Th2-type response during the treatment
period[3_5].
In the present study, we addressed whether Th1 to Th2 immune deviation in IL-12p40-deficient mice could eliminate the
severity of allograft rejection based on the fact that Th1 cells are essential in induced autoimmune
diseases[28] and the observation in
CCR5_/_ mice of an obvious prolongation of islet allograft survival and a switch to Th2
response[18]. We predicted that IL-12p40 might play a determining role in islet allograft rejection in a STZ-induced diabetic mouse model.
Surprisingly, islet allograft from BALB/c mice survived well in IL-12p40- deficient mice (C57BL/6 background) as compared to
wild-type control mice, their mean survival time was similar and the values were 10.0±2.9 d and 9.7±2.1 d, respectively. In
addition, there was no significant difference in the infiltration of CD4+ and CD8+ T cell and the cytokines expression
(IFN-g and IL-4) in transplanted grafts.
According to our observations, although tolerance therapy in some animal models often skewed the immune
activation toward a Th2-dominated response[
26,27], a Th1 to Th2 immune deviation does not uniformly permit the acquisition
of transplant tolerance. This new finding leads us to make an explanation about the mechanisms of allograft
rejection[29,30]. First, we have noticed that the induction of permanent engraftment or a state of allograft tolerance through the administration
of long-acting Th2 cytokines (ie, IL-4-Ig and IL-10-Ig fusion proteins) or immune deviation via the application of IL-12
antagonists have failed in the heart
allograft[30,31]. It is obvious that the strategies, which are extremely effective in dampening
autoimmunity, have not been proven to be effective in
transplantation[32_36]. In accordance with our results, although
anti-IL-12 was used to deviate Th1 to Th2, anti-IL-12 treatment was totally ineffective in prolonging the engraftment of
MHC-mismatched islet allografts. While the impact of anti-IL-12 upon the pattern of cytokine expression was consistent, the
differences were noted in the impact of such therapy upon the duration of engraftment in MHC-mismatched versus
MHC-matched conditions. It is concluded that regarding to the model of Ag presentation and the responding T cell clone number,
the allograft response to MHC-matched rather than minor Ag-mismatched allografts may more closely resemble the response
to auto-antigens[37]. It should be pointed out that a manifestation of a Th2-type response is not equal to an induced Th1 to
Th2 immune deviation manipulation. If they have equal effects
in vivo, logically, the Th2 biased polarization is only to induce
weak tolerance rather than a sufficient condition to block immune response.
The evidence indicates that Th2 cytokine production to either graft survival or rejection is based on assessing intragraft
cytokine gene expression by RT-PCR or identifying graft infiltrating cells that stain for cytokine protein by
immunohistochemistry within allografts. As mentioned by Piccotti
et al[29], these assays do not take into account the antigen specificity
of the cells producing cytokines. Limiting dilution analysis studies have revealed that the frequency of donor-specific T cells
infiltrating grafts is very low. Hence, RT-PCR may detect cytokine mRNA produced by irrelevant cells that are trafficking
through the graft. We also found that the cytokines such as
IFN-g and IL-4 expressed by the grafts had no difference. This
observation suggested that the evaluation of intragraft cytokine profiles for Th1/Th2 dominance should be viewed carefully.
We accepted that all of the cytokine profile reflected a Th2 bias in the graft location, there was no solid evidence to indicate
that Th2 cells are really protective as we had previously thought.
It should be pointed out that IL-12p40 is the common-chain of IL-12 and IL-23. Thus, the IL-12p40-deficient mice have
both cytokines knocked out. Recently, the important role of IL-23 in autoimmune diseases and pathological inflammatory
processes has been unraveled. IL-23 drives the development of a novel T-cell subset characterized by the production of
IL-17 (ThIL-17), which plays a central role in mediating chronic inflammatory
responses[38]. It is not clear whether IL-23 will have
any impact on allograft rejection. Our results suggested that a lack of IL-23 in p40-deficient mice did not inhibit islet
transplantation.
In conclusion, the islet allograft survival is not prolonged in IL-12p40-deficient mice as compared to WT controls. We
conclude that Th cell activation rather than Th1/Th2 polarization plays the major role in controlling allograft rejection. The
precise mechanism for the failure of Th1 to Th2 immune deviation in allograft rejection in MHC-mismatched allograft
recipients will require further investigation.
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