2007) 231–236www.elsevier.com/locate/trim

Transplant Immunology 17 (

Inhibitory effects of anti-CII TA M1-RNA on IFN-γ induced majorhistocompatibility complex class II antigens expression

on cultured human chondrocytes

Fei He a,b, Rong Guo c,⁎, Xin Du c, Ze-Sheng Lu c, Jian-Yu Weng c, Wei Lin c

a Department of Cardiology, Guangdong Provincial People's Hospital, Guangzhou, Guangdong Province, 510080, Chinab Guangdong Cardiovascular Institute, No.96 Dongchuan Road, Guangzhou, Guangdong Province, 510100, China

c Department of Hematology, Guangdong Provincial People's Hospital, No.106 Zhongshan 2nd Road, Guangzhou, Guangdong Province, 510080, China

Received 24 September 2006; received in revised form 23 November 2006; accepted 4 December 2006

Abstract

Major histocompatibility complex class II (MHC-II) trans-activator (CII TA) has been shown to be required for constitutive and IFN-γ-inducedMHC-II transcription. This study investigated the inhibitory effect of anti-CII TA M1-RNA on expression of MHC-II in chondrocytes in responseto IFN-γ. M1-RNAs with different guide sequence (GS) recognizing 452 or 3408 sites in CII TA (M1-452-GS and M1-3408-GS, respectively)were cloned into pUC19 vector. Target mRNA (3176–3560) in CII TA was obtained from Raji cell and inserted into pGEM-7zf(+) plasmid. Therecombinant M1-RNAs and their target mRNA were incubated in a cell-free condition. It showed that only M1-3408-GS could cleave the targetmRNA exclusively. M1-3408-GS was also cloned into psNAV vector (named pA3408). Chondrocytes was stably transfected with pA3408 andexpressions of classical MHC-II (HLA-DR, -DP, -DQ) were analyzed by Flow Cytometry. The level of CII TA mRNAwas measured by RT-PCR.Peripheral blood mono-nucleated cells (PBMNCs) were stimulated by pA3408-positive chondrocytes in mixed lymphocyte reaction, andproliferation of PBMNCs and IL-2 mRNAwere detected. The expression of HLA-DR and HLA-DP on pA3408-positive chondrocytes in responseto IFN-γ decreased 73.00%±5.24%, 88.47%±2.02%, respectively (Pb0.05); So did the content of CII TA mRNA (70.11%±5.79%, Pb0.05).Proliferation of PBMNCs and production of IL-2 mRNA were both inhibited by pA3408 in mixed lymphocyte reaction. This is the firstdescription that anti-CII TA M1-RNA could prevent IFN-γ-induced CII TA transcription and results in a decreased MHC-II expression inchondrocytes.© 2006 Elsevier B.V. All rights reserved.

Keywords: MHC class II; CII TA; Chondrocytes; Ribonuclease P (M1-RNA)

1. Introduction

In tissue engineering, auto-chondrocyte transplantation canbe used to repair isolated cartilage lesions. But auto-chondro-cyte transplantation is not only time-consuming and expensive,but also difficult to be widely spread. Compared with auto-chondrocyte, allo-chondrocytes are a promising source ofcartilage cells with many advantages, including being relativelyplenty source, higher activity of growing into cartilage cells.However, allo-cell transplant rejection associated with thisapproach is still a great challenge.

⁎ Corresponding author. Tel.: +86 20 83827812x62121; fax: +86 2083827712.

E-mail address: gh7311@yahoo.com.cn (R. Guo).

0966-3274/$ - see front matter © 2006 Elsevier B.V. All rights reserved.doi:10.1016/j.trim.2006.12.002

Major histocompatibility complex class II (MHC-II) are α/βheterodimers that participate in the adaptive arm of the immuneresponse by presenting endogenously derived antigenic peptidesto CD4

+ T cells. Although the normal pattern of expression ofMHC-II genes is restricted to antigen presenting cells (APCs),thymic epithelium, and B cells, induction of MHC-II can occuron most cell types through exposure to various cytokines, themost potent of which is interferon-gamma (IFN-γ) [1,2].Expression of MHC-II on cells other than APCs can aid in theinitiation of an acute immune response.

The expression of MHC-II is regulated primarily at the levelof transcription, and class II trans-activator (CII TA) has beendemonstrated as a master control factor for both constitutive andinducible MHC-II expression [3]. CII TA expression patternsparallel to those of MHC-II antigens [4,5]. CII TA-negative cells

Fig. 1. Cleavage of anti-CII TA M1-RNA in vitro. (A) The template RNA of CIITA. pGM-3176 (3176–3560) only included the cutting point of M1-3408-GS,not that of M1-452-GS. (B) Schematic representation of M1-452-GS or M1-3408-GS: GS encoding 12 or 11 nucleotides complementary to CII TA wascovalently linked to the 3′ terminal of M1-452-GS or M1-3408-GS. So M1-452-GS and M1-3408-GS specifically cut CII TA on the site of 452 and 3408respectively. (C) The cleavage of target RNA by M1-RNA in vitro:autoradiograph of transcripts of M1-452-GS (lane 1), M1-3408-GS (lane 2)and target mRNA pGM-3176 (lane 3). pGM-3176 was incubated with M1-3408-GS (lane 4) and with M1-452-GS (lane 5). Expected cleaving stripes—269 nt and 158 nt only appeared in the cleaving products of M1-3408-GSincubated with pGM-3176.

232 F. He et al. / Transplant Immunology 17 (2007) 231–236

are also MHC-II negative. Thus, CII TA expression functions asa molecular switch forMHC-II gene regulation. Inhibition of CIITA expression can prevent embryonic trophoblast cells from up-regulating MHC-II genes in response to IFN-γ. This is thoughtto be one mechanism of maternal tolerance to the fetal allograft[6]. Moreover, the survival of CII TA-deficient cardiac allograftswas prolonged [7]. Therefore, it may be an effective method torepress allo-chondrocyte transplantation rejection by down-regulating the expression of CII TA.

Ribonuclease P is a ribonucleoprotein complex catalyzingthe hydrolysis reaction to remove a 5′ leader sequence from t-RNA precursors [8]. M1-RNA is the catalytic RNA subunit ofRibonuclease P from Escherichia coli and it can be employedas a tool to cleave any specific target sequence, simply byadding a so-called guide sequence (GS) at the 3-terminalcomplementary to the target mRNA and leaving a 5′-ACCAC-3′unpaired stretch (Fig. 1A). Thus, M1-GS RNA can be usedto specifically cleave any target gene complementary to theGS sequence.

2. Objective

The aim of this study was to induce expressions of MHC-IIantigens (HLA-DR, HLA-DP and HLA-DQ) with IFN-γ oncultured human chondrocytes, and then to observe the inhibitoryeffect of anti-CII TA M1-RNA on expression of MHC-II inchondrocytes in response to IFN-γ.

3. Materials and methods

3.1. Plasmids

pTK117, a pUC19 derivative coding M1-RNA, was presented by Dr. BobinChen. pUC19 and pGEM-7zf(+) vectors were purchased from Shanghai SangonCompany, China. Adeno-associated virus vector (psNAV) was presented by Dr.Huazhong Lu.

3.2. Reagents

T4 DNA ligase and restriction endonucleases EcoRI, BglII, BamHI, XhoI,XbaI were purchased from MBI company, USA; mouse fluorescein isothio-cyanate-labelling anti-human isotype control (IgG2a), HLA-DR (IgG2a, k),HLA-DQ (IgG2a, k) monoclonal antibody and recombinant human IFN-γ wereobtained from PharMingen, USA; mouse anti-human HLA-DP (IgG2b, BRA-FB6) was purchased from CYMBUS, mouse anti-human HLA-A,B,C (IgG2a,k) was obtained from LABVISION, while ENETICIN(G418) and TRIZOLR

were done from GIBCO, USA. 2×HiFi Master PCR kit was purchased fromShanghai Sangon Company, China. TITANIUMR one-step RT-PCR kit wasobtained from CLONTECH, USA. DMEM/F12 and RPMI1640 were purchasedfrom GIBCO, USA; while fetal bovine serum was done from HyClone, USA.EndoFreeR Plasmid Maxi Kit and Effectene were purchased from QIAGEN,USA; Ribomax large scale RNA production system-T7 was bought fromPromega, USA. Mitomycin-C was purchased from Sigma, USA.

3.3. Cell culture

Human articular cartilage samples were obtained from patients whounderwent articular replacement. Chondrocytes were isolated from articularcartilage by collagenase II and were grown in 6-well plates with DMEM/F12supplemented with 10% fetal bovine serum [9]. Raji cells (Shanghai Cell Bankof Chinese Academy of Science) were maintained in RPMI1640 supplementedwith 10% fetal bovine serum.

3.4. M1-RNA synthesis

M1-RNA with anti-CII TA GS (M1-452-GS and M1-3408-GS) wasamplified by polymerase chain reaction (PCR) from pTK117 plasmid. Thecommon 5′ primer OliT7: 5′-gcggaattcTAATACGACTCACTATAG-3′,annealing with the T7 promoter and providing a 5′EcoRI site for cloning.The 3′-terminal primers containing the appropriate guide sequences wereOlip3408: 5′-gaagatctGTGGTGCAGCTCGCTGATTACGCCAAGG-3′ andOlip452: 5′-gaagatctGTGGTTCTTCCAGGACTGCCAAGCTTGC-3′. The3′ proximal sequences of 15 nucleotides in Olip3408 and 11 nucleotides inOlip452 served as primers for the PCR with the pTK117 sequence. Thelowercase sequences, the underlined ones and the black ones correspondedrespectively to the SalI in Olip3408 or BglII in Olip452, the ACCAC-3′sequence and GS. The two different PCR products (M1-3408-GS and M1-452-GS) were cloned into pUC19 vector (renamed as pUC19-M1-3408-GSand pUC19-M1-452-GS) (Fig. 1B).

Fig. 2. Expressions of MHC class II antigens in chondrocytes. Values weremean±SD (n=5), ⁎Pb0.05 vs without IFN-γ induction.

233F. He et al. / Transplant Immunology 17 (2007) 231–236

3.5. Construction of CII TA target gene

The target gene in CII TA (3176–3560) was obtained from Raji cell by RT-PCR, according to instructions of TRIZOLR and TITANIUMR one-step RT-PCRkit, which contained the cleavage sites of M1-3408-GS. The 5′ primer was: 5′-ccgctcgagAGCTGAAGTCCTTGGAA-3′; the 3′ primer was: 5′-gcggaattcGAA-CATGCCTGTCCAGAGC-3′. Then the target gene, containing 384 nucleotidesspanning at the point of 3408,was cloned into theXhoI/EcoRI site of pGEM-7zf(+)(renamed as pGM-3176).

3.6. Assays for in vitro transcription and cleavage

Plasmid pGM-3176 was linearized with EcoRI, while pUC19-M1-3408-GSand pUC19-M1-452-GS were linearized with SalI and XbaI respectively.Purification was done using the EndoFreeR Plasmid Maxi Kit. Transcription invitro was performed from the pGEM-7zf or pUC19 T7 promoter according to theinstructions of Ribomax large scale RNAproduction system-T7, then transcribedM1-RNA and their target RNA were mixed and incubated proportionally. Thecleavage products were analyzed on 10% polyacrylamide gels. It showed thatM1-3408-GS could cleave target RNA significantly (Fig. 1C).

3.7. Subclone of M1-3408-GS

To clone M1-3408-GS to the psNAV vector, plasmids pUC19-M1-3408-GSwere digested with EcoRI/SalI. The DNA fragments corresponding to M1-RNA-GS were cut from the gel and purified. These fragments were then ligatedwith the psNAV vector using EcoRI/SalI (psNAV-M1-3408-GS, pA3408). Theauthenticity of pA3408 was confirmed by DNA sequencing.

3.8. Gene transfection by Effectene

Chondrocytes were grown into 6-well (2.5×105 cells/well) plates the daybefore transfection. When chondrocytes could produce 40–80% confluence,they were transfected with pA3408 by Effectene according to the instructions.

3.9. Detection of MHC class II and I expression in chondrocytes

Chondrocytes were collected and reacted with isotype control, anti-humanHLA-DR, HLA-DP, HLA-DQ and MHC class I (HLA-A,B,C) antibodiesrespectively. Death cells were excluded by propidium iodide dye. Theexpression of classical MHC class II and I were detected by Flow Cytometry(COULTER, EPICSXL).

3.10. Detection of CII TA mRNA in chondrocytes by RT-PCR

RT-PCR was performed according to the manual of TRIZOLR andTITANIUMR one-step RT-PCR kit. In a total 50 μl volume, PCR reaction mixturewas firstly incubated for 1 h at 50 °C and 5min at 94 °C, then was run for 30 cyclesof denaturation for 30 s at 94 °C, annealing for 30 s at 65 °C, extension for 1 min at68 °C. Finally, themixture was extended for another 7min at 72 °C after the cycles.To normalize differences in the amount of total RNA added to the reaction,amplification of β-actin was performed as an endogenous control. The optimalcycle number was determined in the preliminary experiments so that the PCRproducts fell in a linear range after amplification. The following primers were used:(1) CII TA sense: 5′-CCGCTCGAGGCTGCCTGGCTGGGATT-3′; CII TAantisense: 5′-GCGGAATTCCGATCACTTCATCTGGTCCTAT-3′ (amplificationlength 410 bp); (2) β-actin sense: 5′-ATCATG TTTGAGACCTTCAA-3′; β-actinantisense: 5′-CATCTCTTGCTCGAAGTC CA-3′ (amplification length 310 bp).Then the PCR products were electrophoresed and separated by 1.5% agarose gelswith ethidium bromide. Quantification of differences in mRNA levels wasperformed using the Eagle EyeII Still Video System (Stratagene, USA).

3.11. Mixed lymphocyte reaction [10]

pA3408-positive chondrocytes (1×107/ml) induced by IFN-γ were firststimulated by mitomycin-C (25 μg/ml), then were incubated for 30 min at 37 °C,

5% CO2 (keeping away light), collected and washed with DMEM/F12 twice, thengrown in 6-well plates (1×106/well) as stimulating cells. Peripheral blood mono-nucleated cells (PBMNC, 1×106) from healthy donors were added into above cellsfor 48 h. The production of cytokine IL-2 mRNA from PBMNC was analyzedthrough RT-PCR. During the last 18 h of culture, [3H] thymidine was added. At theend of incubation, cells were harvested and radioactivity in harvested cells wasmeasured by liquid scintillation counting. The [3H] thymidine incorporation wasexpressed as the median counts per minute of triplicate samples.

3.12. Statistics

Data were presented as mean±SD. Multiple comparisons were evaluated byone-way ANOVA analysis and the significance of the difference between 2groups was analyzed by post hoc test. Values of Pb0.05 were consideredsignificant.

4. Results

4.1. Expressions of MHC class II and I in chondrocytes

All of the expressions of HLA-DR, HLA-DP and HLA-DQ inchondrocytes were low (0.45%±0.27%, 4.17%±1.70% and 0.74%±0.21%, respectively). Induced by IFN-γ (40 ng/ml, 3d), the expressionof HLA-DR and HLA-DP in chondrocytes increased significantly(28.48%±8.11% and 46.20%±4.70%, respectively, Pb0.05). Whilethe expression of HLA-DQ increased mildly upon IFN-γ induction(2.54%±1.32%) (Figs. 2 and 3: representative data from one of theexperiments). The expression of HLA-ABC in chondrocytes did notchange after IFN-γ treatment (40 ng/ml, 3d) (91.40%±8.31%).

4.2. Effect of M1-RNA on MHC-II expressions in chondrocytes

Comparedwith void vector, M1-3408-GS down-regulatedMHC classII expression on pA3408-positive chondrocytes after IFN-γ treatment.The expression of HLA-DR,HLA-DP andHLA-DQdecreased 73.00%±5.24%, 88.47%±2.02% and 44.30%±6.30%, respectively (Pb0.05)(Figs. 4 and 5: representative data from one of the experiments). Theexpression of HLA-ABC on pA3408-positive chondrocytes was notaffected. It was therefore concluded that M1-3408-GS did not inhibitMHC-I expression in chondrocytes after IFN-γ treatment.

4.3. Effect of M1-RNA on expression of CII TA mRNA

Compared with void vector, M1-3408-GS down-regulated expres-sion of CII TA mRNA on pA3408-positive chondrocytes after IFN-γinduction. The induced expression of CII TA mRNA on pA3408-positive chondrocytes decreased 70.11%±5.79% (Pb0.05).

Fig. 5. Comparison of MHC-II expression on pA3408-positive chondrocyteswith that on void-vector chondrocytes after IFN-γ(40 ng/ml, 3d) treatment.p-Chondrocyte: refer to void-vector positive chondrocytes, p3408-Chondro-cyte: refer to pA3408-positive chondrocytes. Live cells were gated andadopted to MHC-II evaluation. The marker line in each panel was setaccording to isotype control (right shows the positive cells or stained withHLA-DR, DP, DQ antibody).

Fig. 3. Comparison of MHC-II expression in chondrocytes with that after IFN-γ(40 ng/ml, 3d) treatment. Chondrocyte: refer to chondrocytes in absence of IFN-γ,Chondrocyte(IFN-γ): refer to chondrocytes upon the induction of IFN-γ. Live cellswere gated and adopted to MHC-II evaluation. The marker line in each panel wasset according to isotype control (right shows the positive cells or stainedwith HLA-DR, DP, DQ antibody).

234 F. He et al. / Transplant Immunology 17 (2007) 231–236

4.4. Mixed lymphocyte reaction

Induced by IFN-γ (30 ng/ml, 3d), void-vector control chondrocytescould cause high expression of IL-2 mRNA in PBMNC while pA3408-positive chondrocytes could hardly induce expression of IL-2 mRNA

Fig. 4. Effect of M1-RNA on MHC-II expression on IFN-γ inducedchondrocytes. Values were mean±SD (n=5), ⁎Pb0.05 vs void vector.

(74.02%±5.01% vs 7.59%±3.11%, Pb0.05). After IFN-γ treatment, ahigher [3H] thymidine incorporation of PBMNC stimulated by void-vector control chondrocytes was obtained as compared with PBMNCstimulated by pA3408-positive chondrocytes ((25.14±1.56)×103 cpmvs (6.34±0.62)×103 cpm, Pb0.05).

5. Discussion

CII TA expression correlates directly with MHC-II expres-sion and is induced by IFN-γ in a time frame that precedesMHC-II gene expression [11,12]. All of the expressions ofHLA-DR, HLA-DP and HLA-DQ in chondrocytes were low(0.45%±0.27%, 4.17%±1.70% and 0.74%±0.21%, respec-tively). Incubation of chondrocytes with IFN-γ caused thestrong induction of HLA-DR and HLA-DP, whereas HLA-DQexpression was largely unaffected. These results are inaccordance with the findings of Lance et al. [13], andcorresponding to the fact that HLA-DR and HLA-DP playmajor roles in transplantation rejection in clinical setting whileHLA-DQ is less important.

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CII TA regulated the transcription of MHC-II gene byinteracting with the trans-acting factors such as RFX, X2BP andNFY [14,15]. The expression of CII TA parallels with that ofMHC-II and CII TA appears only in the MHC-II positive cells[4,5]. Our experiment also showed that MHC-II expressionstringently depended on CII TA: in the absence of IFN-γinduction, chondrocytes hardly expressed CII TA gene andMHC-II antigens; upon IFN-γ induction, the expressions of CIITA gene and MHC-II antigens increased simultaneously.

In order to inhibit the expression of MHC-II, we used M1-RNA to inhibit CII TA gene in our experiment. Ribozymes areof many different kinds such as hammerhead ribozyme, hairpinribozyme, and M1-RNA. Both hammerhead and hairpinribozymes require the GUC sequence to identify in targetsequences [16,17]. However, M1-RNA is not limited by thisrequirement and can aim at any site in target sequence, so it hasa wider selective range [8]. According to human mRNAsequences published in the NCBI Gene Bank, we selected 452and 3408 sites in CII TA gene as target sites for M1-RNA.Proline/serine/threonine-rich protein domain and leucine-richrepeats initiated by these sites are very critical to thetranscription of CII TA gene [18,19]. The GS of M1-3408-GSand M1-452-GS were mapped as 11 and 12 nucleotidesrespectively. The 5′-terminal of M1-RNA had a TAATA box(T7 promoter), which was cloned into the psNAV vector(without T7 promoter). Consequently, there was a transcriptionusing the T7 promoter itself in the M1-RNA. Thus supplemen-tary sequences of the psNAV vector were avoided, objectivelyreflecting the cleaving activity of M1-RNA. Our resultsrevealed the expected cleaving stripes—553 nt and 176 ntappeared only in products of M1-3408-GS and CII TA targetRNA (Fig. 1C). It indicated that only M1-3408-GS could cleaveCII TA target RNA but M1-452-GS does not have this cleavingactivity.

In current study, we successfully constructed pA3408 (orpsNAV-M1-3408-GS) including M1-RNA with anti-CII TAguide sequence. After pA3408 was transfected into chondro-cytes by nanometer Effectene, the induced expression of HLA-DR and HLA-DP on chondrocytes’ surface was lost to a largeextent. Meanwhile, the induced expression of CII TA decreasedsignificantly (70.11%±5.79%). The inhibitory effect of M1-RNA on CII TA and MHC-II antigens seemed still not veryideal. However, in our other experiment, the inhibitory effect ofM1-RNA on CII TA in Hela cells was more than 90%. Wethought that the difference of the results between two studies isrelated to the difference of transfection rate of M1-RNA in thesecells. Future studies will explore a better transfectant forchondrocytes. The reduction of CII TA mRNA was the directreason that the expression of MHC-II resisted the induction ofIFN-γ. Moreover, chondrocytes in response to IFN-γ couldstimulate the expression of IL-2 mRNA in PBMNC, whilepA3408-positive chondrocytes in response to IFN-γ nearly lostthis ability. Therefore, M1-3408-GS could inhibit the family ofMHC-II antigens by repressing CII TA and therefore down-regulate the ability of stimulating mixed lymphocyte reaction.Our results were in accordance with the findings of Luder et al.[14]: TOXO plasma gondi parasite lowered MHC-II expression

in macrophagocyte by inhibiting inducible CII TA mRNAexpression in mouse bone marrow. Moreover, simvastatin andcyclosporine could completely repress MHC-II expression inhuman microvascular endothelial cells by reducing their CII TAmRNA in vitro [20–22].

The inhibition of expression of MHC-II on fetal trophoblastcells is one of the numerous mechanisms that have beenproposed to explain the phenomenon of maternal–fetaltolerance during pregnancy [23–26]. Felix et al. [27] revealeda modest role for surface-expressed donor class II in primedkeratinocyte rejection, but also revealed a dramatic contrast tothe cardiac allograft system. Skin grafts are quite susceptible torejection caused by minor histocompatibility antigen differ-ences [28]. This is in contrast with numerous other organ graftmodels (e.g., heart and kidney), which frequently demonstrateenhanced or indefinite survival of MHC null grafts [29–31].This indicated that the mechanism of graft rejection was likelyto be tissue/organ specific. Minor and class I MHC incompat-ibilities do not cause rejection of heart grafts but influence therejection of skin grafts [30]. RT1.B class II molecules appearedon some chondrocytes in allogeneic transplants and theirexpression increased in the course of cartilage rejection [32].The responses directed against MHC-I and minor histocompat-ibility antigens still have to be elucidated for allo-chondrocytesgraft rejection, and this is our limitation.

In conclusion, this is the first time that M1-RNA has beendemonstrated to down-regulate the expression of CII TA, and itsuggests a way in which one might experimentally control classII for therapeutic intervention. It is regretful that our experi-ments only studied MHC-II in chondrocytes ex vivo and thestudy in vivo is still necessary. Future studies will explorewhether anti-CII TA M1-RNA prolongs the survival ofallogeneic chondrocytes significantly in vivo, or leads to sideeffects, such as severe arthritis and immune suppression.

Moreover, aberrant expression of class II in inappropriatetissues, such as in cases of autoimmune disease (rheumatoid orrheumatic arthritis, sclerosis) [13], can have deleteriousconsequences. It is crucial that the timing and location ofexpression of class II molecules be carefully controlled. Thesefindings revealed the possible applicability of anti-CII TA M1-RNA in potentiating immune tolerance induction for tissuetransplantation and autoimmune disease.

Acknowledgments

We thank Dr. Wang Huizhi and Dr. Lubuser Marshall forcritical reading of the manuscript. This work was supported byGuangdong Provincial Medical Science Research Funds(No. A2005017).

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