Cellular Immunology 272 (2012) 290–292

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Cellular Immunology

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The potential role of preventing atherosclerosis by induction of neonataltolerance to VLDL

Kai Cui a, Guihua Hou b, Yueqiu Feng a, Ting Liang b, Feng Kong c, Lin Sun a, Shumei Wang a,⇑a Department of Epidemiology and Biostatistics, School of Public Health, Shandong University, Jinan, Chinab Department of Radioimmunity, School of Medicine, Shandong University, Jinan, Chinac Department of Biochemistry, School of Medicine, Shandong University, Jinan, China

a r t i c l e i n f o

Article history:Received 24 May 2011Accepted 27 September 2011Available online 8 October 2011

Keywords:Immune toleranceox-LDLVLDLVaccination

0008-8749/$ - see front matter � 2011 Elsevier Inc. Adoi:10.1016/j.cellimm.2011.09.013

⇑ Corresponding author. Address: Department of EpSchool of Public Health, Shandong University, JinanChina. Fax: +86 531 88382141.

E-mail address: cuikai19851022@tom.com (S. Wan

a b s t r a c t

Induction of immune tolerance to ox-LDL could reduce atherosclerosis by modulation immune response.We suppose that very low density lipoprotein (VLDL) may have a similar role to ox-LDL in autoimmuneresponse of atherosclerosis. In this study, neonatal rats were injected with ox-LDL, VLDL, or equal-volumesaline, respectively. Vaccination with ox-LDL reduced the level of specific antibody, T cells proliferationresponse, and the level of endothelins. The method also had a tendency of reducing blood lipids. Vacci-nation with VLDL obviously reduced the level of specific antibody and T cells proliferation. Though therewas also a tendency of reducing blood lipids and endothelins, the effect was less prominent than thatwith ox-LDL. We conclude that, although the effect was less obvious, vaccination with VLDL to induceneonatal tolerance had an effect on modulating immune response, protecting endothelial cells, andreducing blood lipids.

� 2011 Elsevier Inc. All rights reserved.

1. Introduction

Atherosclerosis is considered as an inflammatory disease, andthe involvement of autoimmune response in progression of athero-sclerosis is increasingly recognized [1–6]. At present, considerableattention has been given to oxidized low density lipoprotein (ox-LDL), which is recognized as main autoantigen in initial step of ath-erosclerosis [6–9]. Both of humoral and cell mediated autoimmuneresponse to ox-LDL play an important role in the process of athero-sclerosis [5,10–12]. Along with the development of understandingabout autoimmunity, immune tolerance has been considered as aneffective method for its potential clinical applications for the treat-ment of autoimmune disease [13]. Several studies have indicatedthat induction of tolerance to modulate autoimmune response toox-LDL could reduce atherosclerosis [14,15]. However, there arefew studies about the role of very low density lipoprotein (VLDL)in autoimmune response, which is considered as a native compo-nent of lipoproteins. Accumulating evidence supports that, apoli-poprotein B100 (apo-B100) present in low density lipoprotein(LDL) is modified to form ox-LDL and new epitopes are generatedfor which tolerance is not achieved [7,16]. Since LDL is generatedfrom VLDL by removal of core triglycerides and dissociation of apo-lipoproteins other than apoB-100 [7,17,18], we suppose that it may

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idemiology and Biostatistics,250012, Shandong Province,

g).

play a similar role to ox-LDL in autoimmune response of athero-sclerosis. In our previous study, we had indicated that injectingnewborn rabbits intraperitoneally with VLDL extracted from theserum of hyperlipidemia rabbits could reduce 85% atherosclerosisplaque area and prevent immunopathogenesis of atherosclerosis[19,20]. To further verify our hypothesis, in this study, we vacci-nate the neonatal rats with VLDL to detect whether the methodcan modulate immune response. We also compare the effect ofvaccination with VLDL and ox-LDL on immune response, blood lip-ids, and endothelins.

2. Materials and methods

2.1. Preparation of ox-LDL and VLDL

Forty male Wister rats weighing within 150–180 g were ob-tained from Experimental Animal Center of Shandong University,and were treated in accordance with Animal Protection Act ofChina. After a routine diet for one week, the rats were fed on a cho-lesterol-rich diet (1% cholesterol, 0.2% bile salt, 5% lard oil, and93.8% normal chow) for 30 days, and then their blood was collectedinto sterile centrifuge tubes. The mean serum level of total choles-terol (T-CH) was 5.7 mmol/L, determined by enzyme method kit(Dongou Biotechnology Corporation, Zhejiang, China). LDL andVLDL were extracted from the serum by gradient density centri-fuge. VLDL and LDL were dialysed against PBS, and agarose electro-phoresis was employed to evaluate VLDL and LDL. Copper oxidizedLDL and then ox-LDL and VLDL were respectively filtered by

K. Cui et al. / Cellular Immunology 272 (2012) 290–292 291

0.45 lm pinhead filter (Pall Corporation, USA). After filtration, theoxidation degree of LDL was evaluated with thiobarbituric acidchromatometry, and the result was 4.78 lmol/L. Protein contentof VLDL and ox-LDL after filtration were determined by biuret reac-tion, and the results were 3.80 mg/ml and 2.43 mg/ml respectively.Both of ox-LDL and VLDL were stored at �40 �C.

2.2. Vaccinating with ox-LDL and VLDL in neonatal rats

Male Wister rats, within the first 4 h of life, were randomly di-vided into three groups according to different broods. There were45 rats in ox-LDL group, in which each rat was intraperitoneally in-jected with 0.3 mg ox-LDL; 41 rats in VLDL group, in which each ratwas intraperitoneally injected with 0.3 mg VLDL; 51 rats in controlgroup in which each rat was intraperitoneally injected with equal-volume saline (0.13 ml). 48 h later, some mortality occurred ineach group. Finally, the number of the rats alive in each groupwas 39, 38 and 44 respectively.

2.3. Detection of antibody against ox-LDL and VLDL

All of the above rats had been fed for three weeks until ablacta-tion. Then 10 rats were randomly taken from ox-LDL group, andeach was intraperitoneally injected with 0.3 mg ox-LDL. Ten ratswere randomly obtained from VLDL group and each was intraper-itoneally injected with 0.3 mg VLDL. Twenty rats were randomlyselected from control group, which were randomly divided intotwo groups. Ten rats were intraperitoneally injected with 0.3 mgox-LDL and the other ten were intraperitoneally injected with0.3 mg VLDL. Then the rats were fed on a routine diet for 15 days.At sacrifice, blood was collected and the levels of antibody againstox-LDL or VLDL were detected by an enzyme-linked immunosor-bent assay (ELISA). The results were expressed by the absorbance.

2.4. Establishment of hyperlipoidemia rats models and detection of thelevel of blood lipids and endothelins

Residual rats were fed with a routine diet until they weighedover 150 g, and then fed on a cholesterol-rich diet. Ninety days la-ter, blood was collected and the level of blood lipids and endothe-lins were determined. The serum level of total cholesterol (T-CH)and triglyceride (TG) were determined by an enzyme method kit(Dongou Biotechnology Corporation, Zhejiang, China). The levelof endothelins in serum was determined by a radioimmunoassaykit (Beifang Biotechnology Corporation, Beijing, China). Becausesome rats were sacrificed in preliminary experiment, the numberof remained rats in ox-LDL, VLDL, and control group was 23, 23,and 15 respectively.

Table 1The absorbance of antibody against ox-LDL and VLDL.

Antibody Group Absorbance P

Antibody against ox-LDL ox-LDL group(n = 10) 0.072 ± 0.014 <0.01Control group(n = 10) 0.204 ± 0.025

Antibody against VLDL VLDL group(n = 10) 0.339 ± 0.141 <0.05Control group(n = 10) 0.507 ± 0.021

2.5. Determination of T lymphocytes proliferation response

Blood of 15 rats which were randomly taken out from eachgroup were collected. T lymphocytes were isolated with lympho-cyte isolation liquid (Hengxin Corporation, Shanghai, China) todetermine proliferation response by 3H-thymidine incorporationexperiment. The level of T cells proliferation response was ex-pressed by the counts of per minute (CPM) of 3H-TdR radioactivity.5 � 105 T cells/well were plated in 96-well cell culture plate, andincubated in the following stimulus: ox-LDL (60 ll/ml, dilutedwith RPMI1640-10% FBS), VLDL (30 ll/ml, diluted withRPMI1640–10% FBS), Concanavalin A (2 lg/ml, diluted withRPMI1640-10% FBS) or blank (RPMI1640-10% FBS) respectively.There were three repeated wells in each stimulus, and the CPMof each well was determined for three times.

2.6. Statistical analysis

Results were expressed as means ± SD. Data were analyzed byt-test, one-factor analysis of variance, and Kruskal–Wallisnon-parametric test. Multiple comparisons were analyzed byStudents-Newman–Keuls test. Statistical significance wasconsidered to be P < 0.05.

3. Results

3.1. The level of antibody against ox-LDL and VLDL

To determine the effect of the vaccination on humoral immuneresponse, the level of specific antibody was detected. As shown inTable 1, compared with control group, the specific antibody wasreduced in both VLDL and ox-LDL group.

3.2. T lymphocytes proliferation response to different stimulus

We detected T cells proliferation response in different stimulus,and the results were expressed by CPM. As shown in Table 2, incontrol group, CPM of incubation in ox-LDL was obvious higherthan that in blank, whereas the difference was not significant inox-LDL group. Between ox-LDL and control group, there was no dif-ference in CPM of incubation in either ConA or blank. In VLDLgroup, the difference in CPM between incubation in VLDL andblank was not significant. In control group, CPM of incubation inVLDL was higher than that in blank, but the difference was not sig-nificant. Between VLDL and control group, the difference in the ra-tio of CPM incubation in VLDL to that in blank was significant. Informer group, the ratio was 1.04 ± 0.29, but in the latter was1.26 ± 0.20. Within each group, the CPM of incubation in ConAwas the highest, and that in VLDL group was higher than in theother two groups. But the ratio of ConA to blank had no significantdifference among three groups.

3.3. Blood lipids and endothelins

As shown in Table 3, compared with control group, there was atendency of reducing T-CH, TG and endothelins by vaccinationwith both of ox-LDL and VLDL. However, the difference was notsignificant, except that in the level of endothelins between ox-LDL group and the other two groups. The level of T-CH, TG, andendothelins in ox-LDL group was lower than that in VLDL group.

4. Discussion

In 2000, Nicoletti et al. injected neonatal mice with ox-LDL, anddetected that the vaccination reduced not only immune responseto it, but also susceptibility to atherosclerosis. They also demon-strated the injection induced T-cell tolerance due to clonal dele-tion, rather than anergy of the reactive T cells [14]. We supposedthat VLDL may have the similar role to ox-LDL. As early as 1993,our group detected that vaccinating the rabbits within the first12 h of life with VLDL extracted from hyperlipidemia rabbits couldreduce atherosclerosis plaque area by 85% [19]. In 1995, weindicated that the vaccination could prevent immunopathogenesisof atherosclerosis [20]. However, at that time, the theory of lipo-

Table 2CPM of T lymphocytes proliferation incubation in different medium.

Incubationin

ox-LDL group VLDL group Control group P

ox-LDL 1789 ± 843 — 2441 ± 1074e >0.05VLDL — 2211 ± 985 1729 ± 598 >0.05ConA 23,644 ± 14,118a 47,538 ± 8881c 27,893 ± 10,742f <0.05Blank 1768 ± 1384 2193 ± 556 1485 ± 645 >0.05ox-LDL/

blank1.18 ± 0.42 — 1.79 ± 0.70 <0.05

VLDL/blank — 1.04 ± 0.29 1.26 ± 0.20 <0.05ConA/blank 19.54 ± 11.25b 24.34 ± 10.72d 21.83 ± 7.01g >0.05

in ox-LDL group:a P < 0.01 compared to blank.b P < 0.01 compared to the ratio of ox-LDL to blank;

in VLDL group:c P < 0.01 compared to blank.d P < 0.01 compared to ratio of VLDL to blank;

in control group:e P < 0.05 compared to bland.f P < 0.01 compared to blank.g P < 0.01 compared to ratio of ox-LDL to blank.

Table 3Level of blood lipids and endothelins.

Group T-CH (mmol/L) TG (mmol/L) Endothelins (pg/ml)

ox-LDL(n = 23) 3.30 ± 0.72 0.816 ± 0.231 118.45 ± 44.46a

VLDL(n = 23) 3.35 ± 0.59 0.899 ± 0.348 132.88 ± 38.15Control(n = 15) 3.94 ± 1.22 1.096 ± 0.440 159.96 ± 57.52

a P < 0.05 compared to control group.

292 K. Cui et al. / Cellular Immunology 272 (2012) 290–292

proteins oxidation was not sufficient, and there were several ques-tions remained, such as the suspicion of vaccinating with VLDL,which was considered as native component of lipoproteins. So, inthis study, we compared the effect of vaccination with VLDL andox-LDL on immune response, blood lipids and endothelins.

Our data suggested that vaccination with ox-LDL could modu-late immune response by introduction tolerance in neonatal rats.The data in Table 1 indicated that vaccination with ox-LDL reducedthe humoral immune response to ox-LDL. As shown in Table 2,compared with control group, the method could reduce T cells im-mune response to ox-LDL without affecting T cell proliferation re-sponse to blank and ConA. We also indicated that the methodreduced endothelins, which could be released by injured endothe-lial cells. The data implied that the method could protect endothe-lial cells. Although the difference was not significant, thevaccination had a tendency of reducing blood lipids, and we sup-posed that the method might have an effect on lipid metabolism.

Our data suggested that VLDL might have a similar role to ox-LDL. As shown in Table 1, the humoral immune response was re-duced. The data in Table 2 shown that, between VLDL and controlgroup, there was significant difference in the ratio of CPM incuba-tion in VLDL to that in blank, and the ratio of the former was sig-nificant lower than that in the latter. In control group, the ratioof CPM in ox-LDL to blank was 1.79 ± 0.70, but the ratio of CPMin VLDL to blank was 1.26 ± 0.20. The data implied the vaccinationcould reduce immune response, but the effect was not as obviousas with ox-LDL. In 1993, Li Shaochen of our group vaccinated new-born New Zealand rabbits with VLDL extracted from the serum ofthe rabbits suffering from hyperlipidemia, and significantly re-duced TG and T-CH level, while HLD-CH raised [19]. In this study,the method also had the tendency of reducing the level of bloodlipids and endothelins, but the difference was not significant. Thedifference between conclusions of the two studies may be causedby different animal models, as lipids metabolism differs betweenherbivore and omnivore. Otherwise, the effect of the vaccinationon the level of lipids and endothelins was less prominent than thatwith ox-LDL. Otherwise, in present study, we detected that the

proliferation response to ConA in VLDL group was significantly in-creased, and the response to blank also had a tendency to increase.Further studies were needed to explore the reason for thisimprovement of proliferation response.

VLDL was recognized as a native component of lipoproteins.However, the data both of our previous and present studies indi-cated that vaccination with this lipoprotein could modulate im-mune response by induction of tolerance. In both studies, weextracted the VLDL from the hyperlipidemia animals. So, wesupposed that the VLDL might be modified in the hyperlipidemiaanimals. Vaccination with the VLDL could have a similar effect toox-LDL, but less prominent. If the VLDL was wholly modified tooxidized very low density lipoprotein (ox-VLDL), the vaccinationmight be more efficient.

As a whole, our data demonstrate that vaccination with VLDL inneonatal rats can induce tolerance and may have a potential role ofpreventing atherosclerosis, for its effect on modulating immune re-sponse and protecting endothelial cells, as well as reducing bloodlipids. The findings also prompt that the effect may be more obvi-ous if ox-VLDL is applied in the vaccination.

5. Conflicts of interest statement

No conflicts of interest exist.

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