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Altered Expression of Costimulatory
Molecules in Beh
çet's Disease
According to Clinical Activity
by
Ji Hyun Sim
Major in Medicine
Department of Medical Sciences
The Graduate School, Ajou University
Altered Expression of Costimulatory Molecules
in Beh
çet's Disease According to Clinical Activity
by
Ji Hyun Sim
A Dissertation Submitted to The Graduate School of Ajou University
in Partial Fulfillment of the Requirements for the Degree of
MASTER OF MEDICAL SCIENCES
Supervised by
Eun-So Lee, M.D., Ph.D.
Major in Medicine
Department of Medical Sciences
The Graduate School, Ajou University
This certifies that the dissertation
of Ji Hyun Sim is approved.
SUPERVISORY COMMITTEE
Eun-So Lee
Sun Park
감사의 글
본 논문을 완성할 때까지 지도와 조언을 아끼지 않으셨던 지도교수 이은
소 교수님께 진심으로 감사 드립니다. 또한 좋은 연구를 할 수 있도록 많
은 조언과 격려를 주신 박선 교수님과 손성향 교수님께도 깊은 감사의 마
음을 전합니다. 그리고 연구 기간 동안 도움을 주신 피부 과학 교실원 여
러분과 실험 내내 여러 과정을 도와주신 박미진 선생님과 이미진 선생님,
윤수진 선생님께 감사 드립니다.
언제나 아낌 없는 사랑으로 지원해 주시는 부모님, 가족들에게도 깊은
감사의 마음을 전합니다.
2010년 1월
저자씀
- ABSTRACT –
Altered Expression of Costimulatory Molecules
in Behçet’s Disease According to Clinical Activity
Background: Behçet’s disease (BD) is a multisystemic inflammatory disorder. Several reports have suggested that autoimmunity, more precisely, failure to control autoreactive immune cells in the periphery may play a crucial role in BD. To produce a balanced immune response, key inhibitory costimulartory molecules that critically affect peripheral T cell tolerance and T cell function includes the cytotoxic T-lymphocyte antigen-4 (CTLA-4)/CD80, CD86 and programmed death-1 (PD-1)/programmed death-ligand 1 (PD-L1) receptor/ligand systems are important. Also, regulatory T (Treg) cells play an important role in maintaining immune homeostasis.
Purpose: The objective of the present study was to determine the immunopathological implication of costimulatory molecules in BD. I investigated cell surface expression of CTLA-4 and PD-1 on T cells and Treg cells, as well as cell surface expression of CD80, CD86, PD-L1 on antigen presenting cells (APCs). Also, I measured the concentration of soluble CTLA-4 (sCTLA-4) from serum and culture supernatants.
Materials and Methods: From January 2009 to June 2009, a total of 19 patients with BD were enrolled in this study. The disease control and healthy control (HC) group consisted of
disease and 10 healthy volunteers, respectively. PBMCs of subjects were cultured and stained with the appropriate fluorescent antibody for analysis by flow cytometry. To measure the sCTLA-4 concentrations in serum and in culture supernatants, ELISA was performed. To investigate the mRNA expression level of sCTLA-4 and PD-L1 in PBMCs, real time PCR was performed.
Results: In active BD group, the surface expression of CTLA-4 on CD4+ T cells was decreased in the stimulated state compared with HC group. In contrast, the surface expression of PD-1 on CD4+ T cells and CTLA-4 on CD8+ T cells did not show significant differences among each group. There were no differences of CD80 expression on APCs among each group, however, the surface expression of CD86 on APCs was impaired in active BD group compared with HC and inactive BD group in unstimulated state. Also, the surface expression of PD-L1 on APCs was decreased in active BD group compared with other group in unstimulated state. In addition, the surface expression of PD-L1 on APCs was decreased not only in active BD group, but also in inactive BD group at 48 hours after stimulation compared with other group. Proportion of CD4+CD25+highFOXP3+ T cells on CD4+ T cells was decreased in active BD group compared with inactive BD group. Significantly decreased percentage of CTLA-4 expressing CD4+CD25+high T cells was
observed in active BD group compared with HC group in stimulated state, but surface PD-1 expression on CD4+CD25+high T cells did not show any differences among each group.
Abnormal high serum concentration of sCTLA-4 was appeared in active BD group compared with HC group and RAU group. However, after stimulation, significant differences of sCTLA-4 concentration were not observed in culture supernatants. In
accordance with these results, up-regulated expression of sCTLA-4 mRNA and decreased expression of PD-L1 mRNA in the active BD group were demonstrated compared with the HC group, although it was not statistically significant.
Conclusion: This study shows importance of costimulatory molecules as regulator of balanced immune response in BD. From these results, I suggest that the abnormal expression of costimulatory molecules and serum concentration of sCTLA may have an implication in the pathogenesis of BD.
TABLE OF CONTENTS
ABSTRACT --- i TABLE OF CONTENTS --- iv LIST OF FIGURES --- v LIST OF TABLES --- vi I. INTRODUCTION --- 1II. MATERIALS AND METHODS --- 3
A. Subjects --- 3
B. Methods --- 4
1. Flow cytometry --- 4
2. Cell culture --- 4
3. ELISA --- 5
4. RNA preparation and Real time PCR --- 5
5. Statistical analysis --- 6 III. RESULTS --- 7 IV. DISCUSSION --- 22 V. CONCLUSION --- 29 REFERENCES --- 30 국문요약 --- 38
LIST OF FIGURES
Fig. 1. FACS analysis of CTLA-4, PD-1 on CD4+ T cells and CD4+CD25+high T cells ---- 8
Fig. 2. Surface expression of CTLA-4 and PD-1 on CD4+ T cells (A), proportion of
CTLA-4 (B) and PD-1 (C) expressing T cells on CD4+ T cells --- 9
Fig. 3. Proportion of CTLA-4 expressing T cells on CD8+ T cells --- 11
Fig. 4. Proportion of CD80, CD86, PD-L1 expressing cells on APCs (A to F) --- 13
Fig. 5. Surface expression of CD25 and intracellular expression of FOXP3 on CD4+ T
cells (A), proportion of CD4+CD25+highFOXP3+ T cells on CD4+ T cells (B) ---- 16
Fig. 6. Surface expression of CTLA-4 andPD-1 on CD4+CD25+high T cells(A), proportion
of CTLA-4 (B) and PD-1 (C) expressing T cells on CD4+CD25+high T cells --- 18
Fig. 7. Elevated level of serum sCTLA-4 concentration in active BD group --- 20
LIST OF TABLES
Table 1. Baseline demographics and characteristics of BD patients, RAU patients and healthy control group --- 3
I. INTRODUCTION
Behçet’s disease (BD) is a multisystem inflammatory disorder characterized by recurrent oro-genital ulcers, and cutaneous, ocular, arthritic, gastrointestinal, vascular, and neurovascular involvement. (Sakane et al., 1999) Although the exact pathogenesis of BD is still unclear, several reports have suggested that autoimmunity may play a crucial role. (Mendes et al., 2009)
Autoimmunity in humans is the outcome of a failure to control autoreactive immune cells in the periphery through the modulation of activating and inhibiting signals to produce a balanced immune response. (Fife and Bluestone, 2008) The initiation of T cell activation requires a primary signal delivered by the antigenic peptide presented by major histocompatibility complex (MHC) molecules, and a nonspecific signal generated by the interaction between costimulatory molecules. The costimulatory signal results from the interaction of CD28 on T cells with the B7 family, B7-1 (CD80) and B7-2 (CD86) on antigen presenting cells (APCs). Key inhibitory molecules that critically affect peripheral T cell tolerance and T cell function includes the cytotoxic T-lymphocyte antigen-4 (CTLA-4)/CD80, CD86 and programmed death-1 (PD-1)/programmed death-ligand 1 (PD-L1) receptor/ligand systems. (Fife and Bluestone, 2008; Scalapino and Daikh, 2008)
CTLA-4 is a member of the costimulatory family of molecules for T cells that plays an important role in immunoregulation. (Salomon and Bluestone, 2001; Walunas et al., 1996) CTLA-4 binds CD80 and CD86 with high affinity and competes with CD28. Both CD28 and CTLA-4 bind the same ligands, CD80 and CD86 expressed on APCs, but CTLA-4 has a 20-
PD-1 has been described as a negative regulator of immunity that limits T- and B- cell function. PD-1 is a cell surface molecule expressed on activated T- and B- cells. PD-1 interacts with at least two ligands: L1 and another closely related molecule, L2. PD-1/PD-L1 pathway acts as an important negative regulator of autoimmune response. (Freeman et al., 2000; Kitazawa et al., 2007)
CD4+CD25+highFOXP3+ T cells are a population of regulatory T (Treg) cells which
responsible for active suppression of autoimmunity. (Sakaguchi and Powrie, 2007) Naturally occurring CD4+CD25+highFOXP3+ T cells constitute approximately 10% of peripheral
CD4+ T cells in normal individuals. (Sakaguchi et al., 1995) CD4+CD25+high FOXP3+ T
cells play an important role in maintaining immunological self-tolerance and immune homeostasis. Some reports suggest that CTLA-4 and PD-1 expression on the surface of CD4+CD25+highFOXP3+ T cells has fundamental role in the function of those cells. (Fife and Bluestone, 2008; Kitazawa et al., 2007; Scalapino and Daikh, 2008)
In an attempt to further evaluate the immunopathological implication of costimulatory molecules in BD, I investigated cell surface expression of CTLA-4, 1, CD80, CD86, PD-L1 on peripheral blood mononuclear cells (PBMCs) from BD patients. Also, I measured the concentration of soluble CTLA-4 (sCTLA-4) from serum and culture supernatants. And to clarify the mRNA expression level of costimulatory molecules, real time PCR was performed.
II. MATERIALS AND METHODS
A. SUBJECTS
The patient population consisted of 19 patients with BD (12 women and 7 men; mean ± S.D. age, 35.4 ± 6.5 years), who presented for the first time or were monitored at the Department of Dermatology, Ajou University Hospital from January 2009 to June 2009. Diagnosis was made by BD criteria of either International Study Group or Japanese criteria. (International Study Group for Behçet's Disease, 1990; Lee, 1997) Patients were divided into two groups. Patients in the active group had at least one of the BD symptoms despite treatment and those in the inactive group were in well-controlled states by taking anti-inflammatory medication. The disease control and healthy control (HC) group consisted of age- and sex-matched 8 recurrent aphthous ulcer (RAU) patients without any other evident disease and 10 healthy volunteers, respectively. Informed consent was obtained from patients prior to enrolling them into the study. This study was approved by the Institutional Review Board (IRB number: AJIRB-CRO-08-102).
Table 1. Baseline demographics and characteristics of BD group, RAU group and HC group. Active BD (n=11) Inactive BD (n=8) RAU (n=8) HC (n=10) Age 35.5± 6.6 36.3± 6.1 34.8 ± 9.3 33.7 ± 5.1 Gender Male (%) 4 (36.4%) 3 (37.5%) 3 (37.5%) 4 (40%) Female (%) 7 (63.6%) 5 (62.5%) 5 (62.5%) 6 (60%)
B. METHODS
1. Flow cytometry
PBMCs were prepared from heparinized blood samples by centrifugation over Ficoll Hypaque density gradients (Ficoll paqueTM plus, StemCell Technologies, Vancouver, BC, Canada). For examining cell surface expression of the indicated costimulatory molecules, cells were labeled with the following appropriate conjugated antibodies, APC anti-CD3, FITC CD4, PE-Cy7 CD8, PE-Cy7 anti CD11b, APC-CD11c, PE-Cy7 CD25, PerCP 5.5 CD28, FITC CD80, PE CD86, PE CTLA-4, APC anti-PD-1, PE anti-PD-L1 (BD Biosciences Pharmingen, San Diego, CA, USA), FITC anti-CD3 (eBiosciences, San Diego, CA, USA). For detection of CD4+CD25+highFOXP3+ Treg cells
in the peripheral blood, intracellular FOXP3 staining was carried out on the freshly isolated PBMCs with an intracellular staining kit (eBioscience, San Diego, CA, USA) according to the manufacturer’s instructions. Cell surface and intracellular expression of each molecule was then analyzed by multi-color flow cytometry (BD FACS Canto, San Jose, CA, USA).
2. Cell culture
To evaluate the change of surface expression of costimulatory molecules on PBMC, I performed cell culture and stimulation. PBMCs were cultured in medium (RPMI 1640 medium supplemented with 2mM L-Glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin) with 10% FBS. Cells were stimulated with 25 ng/ml phorbol myristate acetate (PMA) (Sigma, St. Louis, MO, USA) and 1 μg/ml ionomycin (Sigma, St. Louis, MO, USA)
for 48 hrs and stained with the appropriate fluorescent antibody at 24 hours and 48 hours for analysis by flow cytometry.
3. ELISA
To measure the sCTLA-4 concentrations in serum and in culture supernatants (24 hours, 48 hours), ELISA was performed. ELISA kits (MedSystem Diagnostic GmbH, Vienna, Austria) were used for measuring sCTLA-4 levels according to the manufacturer’s protocol and all samples were processed in duplicates.
4. RNA preparation and Real time PCR
Total RNA was extracted using the RNeasy Mini kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. Reverse transcription of RNA was performed using dNTP and oligo(dT) primer (Invitrogen, Carlsbad, CA, USA). The samples were incubated for 5 min at 65°C. The cDNA was then amplified in a 20 μl final volume using SuperscriptTM III (Invitrogen, Carlsbad, CA, USA) following the recommendations of the
manufacturer. SuperscriptTM III contains 5X First-Strand Buffer, 0.1M DTT, RnaseOUTTM
Recombinant Rnase inhibitor, and SuperscriptTM III RT (200 unit/μl). The reaction was
incubated at 55°C for 1 hour, and terminated by 72°C for 15 min and stored at -20°C. Obtained cDNA was analyzed by real-time PCR with the ABI Prism 7000 Sequence Detection System (Applied Biosystems, Foster, CA, USA) according to the protocol provided by the manufacturer and the 2–△△Ct method. I normalized each set of samples using
the difference in threshold cycles (△Ct) between the sample gene and endogeneous control
gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH): △Ct = (△Ctsample –△CtGAPDH).
The calibrator sample (△Ct calibration) was assigned as the sample from HC group. Relative
mRNA levels were calculated by the expression 2–△△Ct, where △△Ct = (△Ctsample –
(Hs01125297_m1) and GAPDH (Hs99999905_m1) were purchased as Assays on Demand primer-probe sets.
5. Statistical analysis
Statistical analysis was performed using SPSS 11.0 software (SPSS Inc., Chicago, IL, U.S.A.). Differences in the expression of costimulatory molecules on PBMCs, and in sCTLA-4 levels in serum and culture supernatants between the groups were analyzed by the Mann-Whitney U test. A value of p<0.05 was considered as statistically significant.
1. Surface expression of CTLA-4 and PD-1 on CD4+ and CD8+ T cells
To evaluate the surface expression of CTLA-4 and PD-1 on freshly isolated cells from patients with BD, PBMCs were stained for surface marker expression with the appropriate conjugated antibodies and analyzed by multi-color flow cytometry. (Fig. 1) In the unstimulated state, there were no differences in the surface expression of CTLA-4 and PD-1 on CD4+ T cells among each group. When PBMCs were stimulated with PMA and ionomycin, increased surface expression of CTLA-4 and PD-1 on CD4+ T cell was observed compared with the unstimulated state. At 24 hours after stimulation, impaired up-regulation of surface CTLA-4 and PD-1 on CD4+ T cells in response to PMA and ionomycin stimulation was observed in all groups. CD4+ T cells from patients in the active BD group expressed a significantly lower level of surface CTLA-4, compared with the cells from the HC group (P<0.05, Fig. 2). On the other hand, no statistically significant differences were observed in the surface expression of PD-1 on CD4+ T cells among each group after the same time period. At 48hrs after stimulation, I could not observe statistically significant differences in surface expression of CTLA-4 and PD-1 on CD4+ T cells among each group. I also evaluated the expression of CTLA-4 on CD8+ T cells but there were no significant differences among each group, both in the unstimulated state and stimulated state (Fig. 3)
(A) 24 hrs PD-1 C T L A -4 HC 0 hrs 48 hrs Active BD Inactive BD RAU
(B)
(C)
Fig. 2. Surface expression of CTLA-4 and PD-1 on CD4+ T cells (A), proportion of CTLA-4 (B) and PD-1 (C) expressing T cells on CD4+ T cells, * p<0.05
2. Surface expression of CD80, CD86, PDL-1 on APC cells
Cell surface expression of CD80 showed no significant differences among each group in both the unstimulated and stimulated state. However, in the unstimulated state, the surface expression of CD86 on APCs was decreased in the active BD group compared with the HC and inactive BD groups (P<0.05). Similarly, PD-L1 expression on APCs was decreased in the active BD group compared with the other group (P<0.05). After stimulation, decreased surface expression of PD-L1 on APCs persisted in the active BD group compared with the other group (P<0.05). At 48 hours after stimulation, decreased surface expression of PD-L1 on APCs appeared in both the active and inactive BD groups compared with the HC group and RAU group (P<0.05). On the contrary, differences in the expression of CD86 disappeared after stimulation (Fig. 4).
(A)
(B)
(D)
(E)
3. Proportion of CD4+CD25+highFOXP3+ T cells on CD4+ T cells and surface
expression of CTLA-4 and PD-1 on CD4+CD25+high T cells
In the active BD group, there were no statistically significant differences in the proportion of CD4+CD25+highFOXP3+ T cells to CD4+ T cells compared with the HC group. However,
significantly elevated proportion of CD4+CD25+highFOXP3+ T cells to CD4+ T cells were
found in the peripheral blood of the active BD group compared with the inactive BD group (P<0.05). On the other hand, no statistically significant differences were observed among other groups (Fig. 5). I also evaluated the surface expression of CTLA-4 and PD-1 on CD4+CD25+high T cells in the unstimulated state and there were no statistically significant
differences of surface expression among each group. After stimulation with PMA and ionomycin, increased surface expression of CTLA-4 and PD-1 on CD4+CD25+high T cells
was observed compared with the unstimulated state. However, no statistically significant differences were observed in the surface PD-1 expression on CD4+CD25+high T cells at 24
and 48 hours among all groups. On the contrary, significantly decreased percentage of CTLA-4 expressing CD4+CD25+high T cells was observed in the active BD group compared
with the HC group at 24 hours after stimulation (P<0.05), but these differences disappeared at 48 hours after stimulation. (Fig. 6)
(A) F O X P 3 HC Inactive BD Active BD RAU CD25 CD25 CD25 CD25 F O X P 3 F O X P 3 F O X P 3 6.9 7.7 4.8 6.8
(B)
Fig. 5 Surface expression of CD25 and intracellular expression of FOXP3 on CD4+ T
(A) 24 hrs PD-1 C T L A -4 HC 0 hrs 48 hrs Active BD Inactive BD RAU
(B)
(C)
Fig. 6. Surface expression of CTLA-4 and PD-1 on CD4+CD25+high T cells (A),
proportion of CTLA-4 (B) and PD-1 (C) expressing T cells on CD4+CD25+high T cells,
4. Elevated level of soluble sCTLA-4 concentration in the active BD group
To evaluate the soluble form of CTLA-4 in human serum, I utilized a sensitive ELISA. Mean serum sCTLA4 concentration of the active BD group (mean ± S.D. = 3.9 ± 1.1) was significantly increased compared with the HC and RAU group (mean ± SD = 2.9 ± 0.4, P< 0.05; Fig. 7). But there were no differences in concentration of sCTLA4 among each group in culture supernatants at 24 hours and 48 hours (Fig. 8).
Fig. 7. Elevated level of serum sCTLA-4 concentration in active BD group (A) (B)
5. mRNA expression levels of sCTLA-4, PD-L1
To clarify the mRNA expression level of sCTLA-4 and PD-L1 which showed significant differences in the active BD group compared with the HC group, the mRNA of the two genes in the PBMCSs was quantified (Fig. 9). Expression of sCTLA-4 mRNA were up-regulated in the active BD group compared with the HC group, although it was not statistically significant (P= 0.21). Decreased expression of PD-L1 mRNA in the active BD group was found compared with the HC group, but without statistical significance (P= 0.15).
(A) (B)
V. DISCUSSION
The immune system has evolved to protect the body from various pathogens but excessive response cause immune mediated tissue damage and autoimmune disease. To accomplish protective role and to avoid the excessive tissue damage, precise regulation of the immune system is needed. This regulation occurs through the modulation of activating and inhibiting signals to maintain a balanced immune response. The initiation of T cell activation requires a primary signal delivered by the antigenic peptide presented by major histocompatibility complex (MHC) molecules, and a nonspecific signal generated by the interaction of co-stimulatory molecules. The coco-stimulatory signal results from the interaction of CD28 on T cells with the CD80 and CD86 on APCs. On the contrary, there are pathways that serve to inhibit T-cell activation, thus play a vital role in maintaining immune homeostasis and these pathways include the CTLA-4/CD80, CD86 and PD-1/PD-L1 receptor/ligand systems. (Fife and Bluestone, 2008; Scalapino and Daikh, 2008)
In current study, the surface expression of CTLA-4 on CD4+ T cells and CD4+CD25+high
cells was decreased at stimulated state in active BD group compared with HC group. In contrast, the surface expression of CTLA-4 on CD8+ T cells and PD-1 expression on CD4+ T cell and CD4+CD25+high showed no statistically significant differences of among each group. The surface expression of CD86 on APCs was impaired in active BD group compared with HC and inactive BD group in unstimulated state. Also, the surface expression of PD-L1 on APCs was decreased in active BD group compared with other group in unstimulated state. In addition, the surface expression of PD-L1 on APCs was decreased not only in active BD
In ELISA, increased serum concentration level of sCTLA-4 was appeared in active BD group compared with HC group and RAU group. Up-regulated expression of sCTLA-4 mRNA and decreased expression of PD-L1 mRNA was observed in active BD group compared with HC group, although it was not statistically insignificant, and these results was consistent with ELISA and FACS analysis.
CTLA-4 is a key molecule that plays an important role in immunoregulation. CTLA-4 appears to inhibit immune responses by several mechanisms and is a critical mediator of peripheral tolerance. (Salomon and Bluestone, 2001; Walunas et al., 1996) CTLA-4 binds CD80 and CD86 with high affinity and competes with CD28. Both CD28 and CTLA-4 bind the same ligands CD80 and CD86 expressed on APCs, but CTLA-4 has a 20- to 50-fold higher affinity than CD28. Therefore, the function of CTLA-4 is to inhibit T-cell activation and proliferation. (Slavik et al., 1999) There are several reports showing reduced expression of CTLA-4 in autoimmune/inflammatory diseases in response to stimulation (e.g., systemic lupus erythematous(SLE), myathenia gravis(MG), Wegener's granulomatosis) (Liu et al., 1998; Steiner et al., 2001; Wang et al., 2002). In accordance with previous reports, data of current study showed that level of increase in surface expression at stimulated state was reduced in active BD group compared with HC group. All these findings support a relation of CTLA-4 expression with autoimmune/inflammatory diseases.
In contrast to cell surface expression of CTLA-4, the plasma concentration of sCTLA-4 was significantly higher in active BD group compared with HC group and RAU group. This is consistent with previous findings of increased levels of sCTLA-4 in patients with SLE, Graves’ disease, and myasthenia gravis. (Liu et al., 2003; Oaks and Hallett, 2000; Wang et al., 2002) Unexpectedly, these results did not correspond with previous study which showed decreased sCTLA-4 concentration in BD group compared with HC group. (Park et al., 2009)
This inconsistence may be appeared because results of previous study were obtained by comparing with BD group, which was not divided to active BD group and inactive BD group, from HC group. Therefore, many number of inactive BD group was included in BD group so that decreased sCTLA-4 concentration in BD group were found compared with HC group.
The effect of elevated sCTLA-4 concentration is unclear. Increased levels of sCTLA-4 may block the interaction between CD80 and CD86 on APCs and CTLA-4 in T cells, thereby interfering with the inhibitory signal sending to T cells, thus the T cell activation would be increased. On the other hand, sCTLA-4 may also block the access of the CD28 on T cells to its ligands, CD80 and CD86, resulting in inhibition of T cell responses. (Liu et al., 2003; Oaks et al., 2000) Therefore, to determine the detailed role of sCTLA-4 in the pathogenesis of BD further investigation would be required. There were no differences of sCTLA-4 concentration in culture supernatants at 24 hours and 48 hours between each group. The expression of the short spliced mRNA, which codes for sCTLA-4 is down-regulated in activated T cells, while it is expressed by resting T cells. Therefore sCTLA-4 plays a more important role in the resting stage of immune response than activated state, because mRNA of sCTLA-4 is constitutively expressed in unstimulated T cells. (Magistrelli et al., 1999)
Genetic variants of the CTLA-4 gene have been found to be associated with susceptibility to several diseases, such as Graves’ disease, SLE, type 1 diabetes and rheumatoid arthritis. (Baniasadi et al., 2006; Fernandez-Blanco et al., 2004; Kula et al., 2006; Yanagawa et al., 2000) CTLA-4 gene polymorphism at position 49 in exon 1 reduces the inhibitory function of CTLA-4 on T-cell proliferation in Graves’ disease. (Kouki et al., 2000) In addition, associated with CTLA-4 gene polymorphism, the role of sCTLA-4 in T cell responses and
there were no any associations between the CTLA-4 49 A/G polymorphism and BD susceptibility, and CTLA-4 -1661 GG genotype was significantly higher in BD than in HC. (Park et al., 2009) Serum sCTLA-4 level in BD patients with the CTLA-4 +49 GG genotype were significantly lower than in HC. (Park et al., 2009) In addition, there were no reports to evaluate the associations between CTLA-4 polymorphism and cell surface expression of CTLA-4. Whether any of these polymorphisms result in alteration of CTLA-4 expression has not been clearly established in BD, therefore correlation of CTLA-4 gene polymorphisms and cell surface expression of CTLA-4 and sCTLA-4 would give a comprehension of genetic association in BD.
Cell surface expression of CD80 showed no significant differences among each group both unstimulated and stimulated state. However, in unstimulated state, the surface expression of CD86 on APCs was decreased in active BD group compared with HC and inactive BD group. APCs especially dendritic cells and macrophages are important in early interactions with T cells and they can have profound effects on the immune response. When either CD80 or CD86 bind to the CD28 receptor, a costimulatory signal is produced that result in activation and proliferation of T cells. In contrast, binding of either CD80 or CD86 to CTLA-4 appears to produce inhibit signals which results in T cell anergy. (Scalapino and Daikh, 2008) Previous studies showed different functions of CD80 and CD86. For example, experimental allergic encephalomyelitis model of mice, treatment with anti-CD80 monoclonal antibody reduced disease severity, however, anti-CD86 monoclonal antibody worsened the disease. (Racke et al., 1995) On the contrary, treatment of NOD mice with anti-CD80 monoclonal antibody exacerbated the spontaneous diabetes, whereas anti-CD86 monoclonal antibody had protective role. (Lenschow et al., 1995) Therefore further study to determine the implication of CD80 and CD86 in BD should be needed.
PD-1 and its ligands, PD-L1 and PD-L2, have been shown to play an important role in regulating T cell activation and maintaining immune homeostasis. PD-1 is an inhibitory costimulatory receptor expressed on activated T, B, and myeloid cells. (Freeman et al., 2000; Kitazawa et al., 2007) In mice model, PD-1-deficieny leads to splenomegaly, a lupuslike glomerulonephritis, and destructive arthritis. (Nishimura et al., 1999) Also, diabetes and multiple sclerosis was induced by using blocking reagents of 1. (Ansari et al., 2003) PD-L1 is expressed on resting T cells, B cells, dendritic cells (DC), and macrophages. (Liang et al., 2003) Blockade or deficiency of PD-L1 in mice results in accelerated disease of diabetes and autoimmune hepatitis. (Dong et al., 2004; Guleria et al., 2007) In the results of present study showed no statistically significant differences of PD-1 expression on CD4+ T cell among each group, in contrast, the surface expression of PD-L1 on APCs was decreased in active BD patients compared with other group. Also, at 48 hours after stimulation decreased expression of PD-L1 was appeared in inactive BD group compared with HC group and RAU group. Previous reports suggest that the PD-1/PD-L1 pathway acts as an important negative regulator of autoimmune response. In this study, these results suggest abnormal PD-1/PD-L1 pathway caused by impaired PD-L1 expression would be important pathogenic mechanism in BD. Interestingly, expression of PD-L1 was decreased not only in active BD group, but also in inactive BD group at 48hrs after stimulation and, until 48 hours, decreased expression of PD-L1 in inactive BD group was not revealed. Some authors suggest that CTLA-4/B7 pathway plays a dominant role during tolerance induction, while PD-1/PD-L1 pathway plays the dominant role during the maintenance of tolerance. (Fife and Bluestone, 2008) And results of this study correspond with these suggestions and future work should focus on the
suppression of autoimmunity. Naturally occurring CD4+CD25+highFOXP3+ T cells
constitute approximately 10% of peripheral CD4+ T cells in normal individuals. (Sakaguchi and Powrie, 2007) This study showed significantly elevated proportion of CD4+CD25+high
FOXP3+ T cells in CD4+ T cells were found in the peripheral blood of patients in active BD group compared with inactive BD group. Furthermore, significantly decreased percentage of CTLA-4 expressing CD4+ CD25+high T cell was observed in active BD group compared with
HC group at stimulated state. Results of the present study consistent with the results of earlier studies which also reported that active BD group had significantly higher CD4+CD25+highFOXP3+ T cells, as compared with inactive BD group. (Hamzaoui et al.,
2006) Significantly higher number of CD4+CD25+highFOXP3+ T cells was observed in
cerebral spinal fluid of BD and multiple sclerosis and peripheral blood of Sjögren syndrome. (Hamzaoui et al., 2007) However, in some autoimmune diseases such as SLE, Kawasaki disease, juvenile idiopathic arthritis, reduced levels of Treg cells were described. (de Kleer et al., 2004; Furuno et al., 2004; Liu et al., 2004) Also, lower amounts of Treg cells correlated with disease activity or poor prognosis in Kawasaki disease patients. (Furuno et al., 2004) The function of CTLA-4 in the Treg cells has been similarly difficult to establish. Treg cells are characterized by their persistent expression of CTLA-4 on their surface. Treg cells which express higher levels of surface CTLA-4 appeared to have higher inhibitory function in vitro. (Liu et al., 2006; Sansom and Walker, 2006) On the contrary, CTLA-4 knockout mice produce Treg cells expressing FOXP3 that has suppressive function in vitro. (Tang et al., 2004) Although CTLA-4 does not seem to be needed for the development or function of Treg cells absolutely, it has significant role in the suppressive function of Treg cells. (Scalapino and Daikh, 2008) One possible explain of increased number may be that Treg cells modulate the immune response and increased number of Treg cells would be induced to
suppress activated state. Also, functional deficiency Treg cells in multiple sclerosis, rheumatoid arthritis was reported, therefore, these results suggest that increased number of dysfunctional Treg cells, may be due to decreased expression of CTLA-4, would have an implication on the development of symptom in BD. (Hamzaoui, 2007)
V. CONCLUSION
This study provides importance of costimulatory molecule as regulator of balanced immune response. In active BD group, the surface expression of CTLA-4 on CD4+ T cells and CD4+CD25+high cells was decreased at stimulated state compared with HC group. The surface expression of CD86 on APCs was impaired in active BD group compared with HC and inactive BD group in unstimulated state. Also, the surface expression of PD-L1 on APCs was decreased in active BD group compared with other group. In addition, the surface expression of PD-L1 on APCs was decreased not only in active BD group, but also in inactive BD group at 48 hours after stimulation compared with other group. Furthermore, abnormal serum concentration of sCTLA-4 was appeared in active BD group compared with HC group and RAU group. Therefore, from these results, I suggest that the abnormal expression of costimulatory molecules and serum concentration of sCTLA may interfere the inhibitory signals which disturb the proper regulation of immune homeostasis, so that have an implication in the pathogenesis of BD.
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- 국문요약 -
베체트병에서 임상 증상의 활성도에 따른
costimulatory molecules의 발현 변화
아주대학교 대학원 의학과 심 지 현 (지도교수: 이 은 소) 연구 배경: 베체트병은 점막 및 피부, 눈, 혈관, 신경 및 기타 여러 장기를 침범 할 수 있는 임상적 특징을 가지는 만성, 재발성의 전신질환이다. 현재까지 베체 트병에서 보이는 지속적인 염증 반응의 정확한 원인 기전은 밝혀져 있지 않으나 면역반응의 조절 이상으로 인한 자가면역이 중요한 기전으로 생각되고 있다. 면 역반응의 조절을 위해 활성화된 T 세포의 억제가 중요 하며 이를 담당하는 주된 기전 중 하나로 costimulatory molecules인 cytotoxic T-lymphocyte antigen-4 (CTLA-4)/CD80, CD86 그리고 programmed death-1 (PD-1)/programmed death-ligand 1 (PD-L1)의 경로를 통한 억제 작용이 있다. 또한 조절 T 세포 역시도 이러한 면역반응의 조절에 중요한 역할을 하고 있다. 연구 목적: 본 연구에서는 베체트병의 병인기전에서 costimulatory molecules의와 PD-1의 발현 정도 및 항원 전달 세포에서의 CD80, CD86 과 PD-L1의 발현 정도를 측정하였다. 또한 환자의 혈장 및 배양액에서의 sCTLA-4의 농도를 측정하였고, 실험 대상 혈액에서의 mRNA 발현의 정량화를 위해 real time PCR을 시행하였다. 연구 방법: 2009년 1월부터 6월까지 내원한 베체트병 환자 중 19명을 대상으로 하였다. 또한 나이 및 성별을 맞춘 재발성 아프타성 구강 궤양 환자 8명을 질환 대조군으로, 10명의 건강한 성인을 정상 대조군으로 선정하였다. 모집된 각 그룹으로부터 얻은 혈액에서 분리된 PBMC를 자극 및 배양 하였고, 세포표면의 costimulatory molecules의 발현 측정을 위해 multi-color flow cytometry, 혈장 및 배양액의 sCTLA-4의 농도 측정을 위해 ELISA, 실험 대상 혈액에서의 mRNA 발현의 정량화를 위해 real time PCR을 시행하였다.
연구 결과: 활동성 베체트병 환자군에서 CD4+ T 세포 및 조절 T 세포 표면에서의 CTLA-4의 발현이 자극 후 24시간 상태에서 정상과 비교하여 낮은 경향을 보였다. 반면에 CD4+ T 세포 및 조절 T 세포 표면에서의 PD-1의 발현은 각 군간에 유의한 차이를 보이지 않았다. CD8+ T 세포 표면에서의 CTLA-4의 발현 역시 각 군간에 유의한 차이를 보이지 않았다. 항원 전달세포에서 CD80의 발현은 각 군간에 유의한 차이을 보이지 않았으나, CD86의 경우 활동성 베체트병 환자군에서 정상 및 재발성 아프타성 구강 궤양 환자군에 비해 낮은 발현을 보였고 PD-L1의 발현은 활동성 베체트병 환자군에서 다른 군들과 비교하여 낮은 경향을 보였다. 또한, PD-L1의 경우 비활동성 베체트병 환자군에서도 자극 후 48시간에서 정상 및 재발성 아프타성
구강 궤양 환자군과 비교하여 발현이 낮은 경향을 관찰할 수 있었다. 혈장에서의 sCTLA-4 농도는 정상 및 재발성 아프타성 구강 궤양 환자군과 비교하여 활동성 베체트병 환자군에서 유의하고 높은 결과를 보였다. 반면에 배양액에서의 sCTLA-4 농도는 각 군간에 차이를 보이지 않았다. 결론: 본 연구는 베체트병 환자군에서 면역 반응 조절 인자로서 costimulatory molecule의 역할의 중요성을 확인 시켜 주었다. 본 연구의 결과들은 비정상적인 costimulatory molecule의 발현과 혈장 에서의 높은 sCTLA-4 농도가 베체트병의 증상 발생에 중요한 역할을 한다는 것을 제시하였다.
