Cellular Immunology 290 (2014) 226–232

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Cellular Immunology

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Effects of TLR4 on b2-glycoprotein I-induced bone marrow-deriveddendritic cells maturation

http://dx.doi.org/10.1016/j.cellimm.2014.07.0060008-8749/� 2014 Elsevier Inc. All rights reserved.

Abbreviations: APS, antiphospholipid syndrome; aPL, antiphospholipid antibod-ies; b2GPI, b2-glycoprotein I; anti-b2GPI/b2GPI, anti-b2-glycoprotein I/b2-glyco-protein I; TF, tissue factor; TLR4, Toll-like receptor 4; TLRs, Toll-like receptors; DCs,dendritic cells; APCs, antigen-presenting cells; MHC, major histocompatibilitycomplex; BMDCs, bone marrow-derived dendritic cells; FBS, fetal bovine serum;GM-CSF, granulocyte macrophage colony stimulating factor; IL-4, interleukin 4;iDCs, immature dendritic cells; mDCs, mature dendritic cells; aCL, anticardiolipinantibodies; anti-b2GPI, anti-b2-glycoprotein I antibodies; LA, lupus anticoagulant;MLR, mixed leukocyte reaction.⇑ Corresponding author at: Department of Clinical Laboratory and Hematology,

School of Medical Science and Laboratory Medicine of Jiangsu University, Zhenjiang,Jiangsu 212013, PR China. Fax: +86 511 85038449.

E-mail addresses: hongzhou@ujs.edu.cn (H. Zhou), yanjinchuan@hotmail.com(J. Yan).

1 These authors contributed equally to this work. / Joint first authors.2 Co-correspondence.

Jingjing Liu a,b,1, Chao He b,1, Hong Zhou a,b,⇑, Ya Xu b, Xiaolei Zhang b, Jinchuan Yan a,2, Hongxiang Xie b,Si Cheng b

a Department of Cardiology, Affiliated Hospital of Jiangsu University, Zhenjiang, Jiangsu 212013, PR Chinab Department of Clinical Laboratory and Hematology, School of Medical Science and Laboratory Medicine of Jiangsu University, Zhenjiang, Jiangsu 212013, PR China

a r t i c l e i n f o

Article history:Received 24 February 2014Accepted 23 July 2014Available online 1 August 2014

Keywords:Dendritic cellsb2-Glycoprotein ITLR4

a b s t r a c t

Our previous study has demonstrated that Toll-like receptor 4 (TLR4) can contribute to anti-b2-glycopro-tein I/b2-glycoprotein I (anti-b2GPI/b2GPI)-induced tissue factor (TF) expression in human acute mono-cytic leukemia cell line THP-1. However, the role of TLR4 in the activation of autoimmune response inantiphospholipid syndrome (APS) has rarely been reported. In this study, we focused on the role ofTLR4 in b2GPI-induced maturation of bone marrow-derived dendritic cells (BMDCs). iDCs from C3H/HeN mice stimulated with b2GPI were more mature than that from C3H/HeJ mice, yields of CD11c+

MHCII+DCs, CD11c+CD80+DCs and CD11c+CD86+DCs and production of some pro-inflammatory cytokinesin iDCs from C3H/HeN were higher than those from C3H/HeJ (p < 0.05). Moreover, the ability of b2GPI-treated iDCs from C3H/HeJ to stimulate proliferation of allogeneic mixed lymphocytes was lower thanthat of iDCs from C3H/HeN. In conclusion, our results indicate that TLR4 may play a significant role inb2-glycoprotein I-induced BMDCs maturation.

� 2014 Elsevier Inc. All rights reserved.

1. Introduction

Antiphospholipid syndrome (APS) is an autoimmune diseasethat is related to high titer antiphospholipid autoantibodies (aPL)and characterized by arterial/venous thrombosis and recurrentfetal loss [1]. The presence of circulating pathogenic aPL is a keyto the diagnosis of APS [2]. Meanwhile, b2-glycoprotein I (b2GPI),as the major target of aPL, can bind to cell surface receptors and

activate various cells, including endothelial cells and monocytes[3,4]. Our previous study has demonstrated that Toll-like receptor4 (TLR4) plays an important role in the process of b2GPI-inducedtissue factor (TF) expression in monocytes, which could contributeto blood coagulation [5].

The mammalian Toll-like receptors (TLRs) are a family of inte-gral membrane protein receptors expressed by immune cells,including macrophages, dendritic cells (DCs), neutrophils, andendothelial cells [5,6]. TLR signaling transduction can result ininnate immune activation and inflammatory response [7]. It wasalso reported by Raschi et al. that TLR serve as sentinels to induceprompt inflammatory and to mediate activation in effector cells ofimmune system [8]. TLR4 is the first member of the pattern recog-nition receptor family and identified as a critical receptor in thehost defense against infection [9]. Previous studies have demon-strated that TLR4 can be activated by several proteins and LPS isthe natural ligand of TLR4 [10–12].

Dendritic cells (DCs) are specialized antigen-presenting cells(APCs) that play a fundamental role in host innate and adaptiveimmunity [10,13–15]. DCs maturation is a fundamental checkpointin the initiation and regulation of immune responses [16]. Imma-ture dendritic cells (iDCs) act as sentinels and can be present inalmost all tissues, where they capture and process antigens. Once

J. Liu et al. / Cellular Immunology 290 (2014) 226–232 227

stimulated by foreign antigens, the iDCs will become mature den-dritic cells (mDCs) after migrating to the T-cell-dependent areas.Meanwhile, the stimulation will enhance their capacity to captureantigen, process it and load peptides onto major histocompatibilitycomplex (MHC) molecules, then transporting them to the cell sur-face [17–19]. Naïve T cells could be readily stimulated with thehigh and constitutive expression of co-stimulatory molecules, suchas CD80 and CD86 on DCs surface [20]. During DCs maturation,MHC, co-stimulatory and adhesion molecules on the cell surfaceare up-regulated and the production of cytokines is increased[17]. The maturation of DCs is important to their immunostimula-tory ability in innate and adaptive immunity [21].

Though many cell types express TLR, DCs are one of the mosteffective responders [14]. It has been revealed that pro-inflamma-tory mediators will be released after TLR4 activated on dendriticcells. With these mediators, innate immune response and adaptiveimmunity are rapidly activated, eventually leading to generation ofantibody [17,22]. It has been discovered that a commercial prepa-ration of human b2GPI could stimulate macrophages to produceTNF-a in a TLR4-dependent manner [23].

Despite previous studies have investigated TLR4-mediated sig-naling pathways in APS, the roles of TLR4 in the activation of auto-immune reaction and the production of corresponding antibodiesof APS have not been completely investigated. In this study, wespotlighted whether TLR4 plays an important role in the processof b2GPI-induced maturation of bone marrow-derived DCs(BMDCs) using two types of mice model (C3H/HeN, TLR4 intactand C3H/HeJ, TLR4 defective).

2. Materials and methods

2.1. Reagents

RPMI-1640 medium was purchased from Gibco BRL (GrandIsland, NY, USA). Fetal bovine serum (FBS) was from Hyclone(Logan, UT, USA). Recombinant murine granulocyte macrophagecolony stimulating factor (GM-CSF) was purchased from HangzhouClongene Biotech Co., Ltd. (Zhejiang, China). Interleukin (IL)-4 waspurchased from Peprotech (Rocky Hill, USA). LPS was from Sigma(Escherichia coli, strain 0128:B12). b2GPI was obtained from USBiological (Swampscott, MC, USA) and its purity is greater than95% checked by SDS–PAGE. FITC-conjugated anti-CD11c, PE-conjugated anti-MHC class II, PE-conjugated anti-CD80 or CD86and their isotype controls were purchased from eBioscience (SanDiego, CA, USA). Mouse IL-12, TNF-a and IL-1b enzyme-linkedimmunosorbent assay (ELISA) kits were from Sunny ELISA Kits(Multisciences, Hangzhou, China). Percoll was from TBD sciences(Tianjin, China). Misogynic C was purchased from Roche (Mann-heim, Germany). CellTiter 96� AQueous One Solution Reagentwas purchased from Promega (WI, Madison, USA). All the reagents(except for LPS) used in our assays were subjected to Detoxi-Gel™(Pierce, Rockford, IL, USA) to remove endotoxin contamination(<0.03 EU/ml) by Limulus amebocyte lysate assay (ACC, Falmouth,MA, USA).

2.2. Mice

Male C3H/HeJ mice (8–10 weeks old) were purchased fromModel Animal Research Center of Nanjing University, PR China.Male C3H/HeN mice (8–10 weeks old) were purchased from VitalRiver Laboratory Animal Technology Co. Ltd. of Beijing, PR China.Mice of C3H/HeJ strain have a missense mutation in the third exonof the Toll-like receptor-4 gene (Tlr4) and a defective response tobacterial endotoxin. In contrast to C3H/HeJ mice, the substrainsC3H/HeN, which diverged from the same stock as C3H/HeJ mice,exhibit vigorous responses to LPS [24]. Male BALB/c mice

(8–10 weeks old) were purchased from the Animal Research Cen-ter of Yangzhou University, PR China.

2.3. Bone marrow preparation

All procedures were conducted under sterile conditions, theanimal experiments were approved by the Laboratory AnimalAdministration Committee of Jiangsu University and performedaccording to the Guide for the Care and Use of Laboratory Animalspublished by the US National Institutes of Health. The femurs ofmale C3H/HeN or C3H/HeJ mice were dissected and separated fromthe surrounding muscle tissue with fine scissors and tweezers. Theintact bones were placed in a dish with 75% alcohol for 1 min,washed twice with phosphate buffered saline (PBS) and movedinto a fresh dish with PBS. Next, both ends of the bones werepierced with a 1 ml syringe, the marrow was flushed out withRPMI-1640 Medium supplemented with 1% glutamine and 1% pen-icillin/streptomycin using the syringe. The clusters in the marrowsuspension were disrupted by vigorous pipetting, and collectedby centrifugation at 1500 rpm for 10 min. Erythrocytes were lysedwith ammonium chloride (8.29 mg/ml NH4Cl, 1.0 mg/ml KHCO3,37.2 lg/ml EDTA-Na2). Finally, the cells were washed once inRPMI-1640 medium.

2.4. Bone marrow-derived dendritic cells (BMDC)

Bone marrow cells were placed in 6-well plates (2 � 106/well,Corning, Corning, NY) in murine DCs media composed of RPMI-1640 Medium supplemented with 1% glutamine, 1% penicillin/streptomycin, 10% fetal bovine serum (FBS), 10 ng/ml recombinantmurine GM-CSF and 10 ng/ml IL-4. After incubation for 48 h, themedium containing non-adherent cells was removed by gentlyswirling the plates and replaced with fresh medium as describedabove. Then half of the media was exchanged every other day.On day 6, iDCs, loosely adherent and nonadherent cells, were har-vested by gentle pipetting and washed once with PBS. iDCs werere-suspended at 1 � 106/ml in murine DCs media containing5 ng/ml GM-CSF and 5 ng/ml IL-4 and plated on 6-well plates at2 ml/well. After stimulation with 1 lg/ml LPS or 50 lg/ml b2GPI[the concentration of reagents is based on our preliminary study,data not shown] for 12 h, the morphological changes of cells wereobserved under inverted phase contrast microscope (Olympus,Tokyo, Japan) every day. 1 lg/ml LPS was included in the assayas positive control. For negative control, the cells were culturedin medium only. DCs were harvested and used for phenotypic anal-ysis or functional assays.

2.5. FACS analysis

DCs treated with 1 lg/ml LPS or 50 lg/ml b2GPI for 12 h werewashed once with PBS and stained with FITC-conjugated anti-CD11c and PE-conjugated anti-MHC class II, PE-conjugated anti-CD80 or CD86 or their isotype controls at 4 �C for 30 min. Cellswere then washed twice and resuspended in 250 ll PBS for flowcytometry analysis (FACSCalibur, BD) and the data were analyzedusing CellQuest (BD Biosciences) software.

2.6. Cytokine assay

Supernatants of DCs from C3H/HeN or C3H/HeJ mice stimulatedwith 1 lg/ml LPS or 50 lg/ml b2GPI for 12 h were collected andstored at �70 �C pending analysis. Concentrations of the cytokinesIL-12, TNF-a and IL-1b were measured using commercially avail-able enzyme-linked immunosorbent assay (ELISA) kits accordingto the manufacturer’s instructions.

Fig. 1. Morphology changes of iDCs from C3H/HeN or C3H/HeJ BMDC induced byb2GPI iDCs from C3H/HeN or C3H/HeJ mice were treated with LPS (1 lg/ml) orb2GPI (50 lg/ml) for 12 h. Those cells were then observed under inverted phasecontrast microscope (�400). Activated BMDC from C3H/HeN mice exhibited longerdendritic protrusions, which is a characteristic of mature DCs. Activated BMDC fromC3H/HeJ mice showed fewer dendrites.

228 J. Liu et al. / Cellular Immunology 290 (2014) 226–232

2.7. Lymphocyte preparation/T cell proliferation assay

T cells were isolated from BALB/c spleen cells by Percoll accord-ing to the manufacturer’s instructions. In triplicate, T cells wereplated at 2 � 105/well in 96-well plates (Corning, Corning, NY)together with 1 � 104 misogynic C-treated (25 lg/ml, 30 min,37 �C) BMDCs per well. On day 3, CellTiter 96� AQueous One Solu-tion Reagent was added into wells of cultured cells (40 ll/well) andthen incubated at 37 �C and 5% CO2 in a humidified incubator for4 h. Finally, color development was monitored by the absorbanceat 490 nm using a kinetic microplate reader (Gene Company Lim-ited, Hong Kong, China).

2.8. Statistical analysis

For all experiments, at least triplicate determinations weremade for each experimental condition. In indicated cases, theresults of representative experiments were shown. Normally dis-

Fig. 2. FACS analysis the expression of CD11c and MHCII molecules on DCs surface iDCs(50 lg/ml) for 12 h, stained with antibodies against the dendritic cell subset markers CDlines (solid lines). The data are representative and pooled date from three separate expe

tributed data were expressed as means ± SEM. Three or moremeans were assessed by a one-way ANOVA. Two-factor treatmentresults were analyzed by two-way ANOVA. Data analyses wereperformed using SPSS statistical software package version 17.0.Differences in data values were define significant at p < 0.05.

3. Results

3.1. Morphology changes of iDCs from C3H/HeN or C3H/HeJ bonemarrow-derived dendritic cells induced by b2GPI

It has been demonstrated that the morphology is differentbetween immature dendritic cells (iDCs) and mature dendritic cells(mDCs) [25,26]. In this study, we observed the different morphol-ogy on b2GPI-treated DCs from C3H/HeN and C3H/HeJ mice. Asshown in Fig. 1, after stimulation with LPS (as a positive control)or b2GPI, a large number of BMDCs from C3H/HeN mice grew insuspension status and showed irregular and long dendritic protru-sions compared with untreated BMDCs, while BMDCs from C3H/HeJ mice appeared to be adherent and showed fewer and shorterdendritic protrusions. These data suggest that BMDCs from C3H/HeN mice are more mature than those from C3H/HeJ mice inresponse to b2GPI.

3.2. Expression of CD11c, MHCII and costimulatory molecules inBMDCs from C3H/HeN or C3H/HeJ mice after stimulation with b2GPI

To demonstrate the roles of TLR4 in the process of mouseBMDCs maturation, the BMDCs from C3H/HeN or C3H/HeJ micewere stimulated with 1 lg/ml LPS or 50 lg/ml b2GPI for 12 h,the expression of CD11c, MHCII and costimulatory molecules(CD80 and CD86) on cells were examined. As shown in Fig. 2, thepercentages of CD11c+MHCII+DCs from C3H/HeN mice were signif-icantly higher than those from C3H/HeJ mice after LPS (75.54% vs.48.15%) and b2GPI (74.38% vs. 50.73%) stimulation. Furthermore,LPS or b2GPI stimulation could obviously increase the expressionof CD80 and CD86 on BMDCs from C3H/HeN mice but not fromC3H/HeJ mice (Figs. 3 and 4).

(2 � 106) from C3H/HeN or C3H/HeJ mice were treated with LPS (1 lg/ml) or b2GPI11c and MHCII, and analyzed by FACS. Isotype controls were used to set quadrantriments yielding similar results.

Fig. 3. FACS analysis the expression of CD11c and CD80 molecules on DCs surface iDCs (2 � 106) from C3H/HeN or C3H/HeJ mice were treated with LPS (1 lg/ml) or b2GPI(50 lg/ml) for 12 h, stained with antibodies against the dendritic cell subset markers CD11c and CD80, and analyzed by FACS. Isotype controls were used to set quadrant lines(solid lines). The data are representative and pooled date from three separate experiments yielding similar results.

Fig. 4. FACS analysis the expression of CD11c and CD86 molecules on DCs surface iDCs (2 � 106) from C3H/HeN or C3H/HeJ mice were treated with LPS (1 lg/ml) or b2GPI(50 lg/ml) for 12 h, stained with antibodies against the dendritic cell subset markers CD11c and CD86, and analyzed by FACS. Isotype controls were used to set quadrant lines(solid lines). The data are representative and pooled date from three separate experiments yielding similar results.

J. Liu et al. / Cellular Immunology 290 (2014) 226–232 229

3.3. Cytokine secretion by BMDCs from C3H/HeN or C3H/HeJ mice inresponse to b2GPI

To further illustrate the role of TLR4 in BMDCs maturation,supernatant of DCs from C3H/HeN and C3H/HeJ mice was analyzedby ELISA. As shown in Fig. 5A, compared with untreated DCs fromC3H/HeN mice or LPS- or b2GPI-treated DCs from C3H/HeJ mice,the DCs from C3H/HeN mice produced significantly higher levelof IL-12 after either LPS or b2GPI stimulation (p < 0.05). The level

of TNF-a in LPS- and b2GPI-treated DCs from C3H/HeN mice wasabout 6- and 8-fold higher than that in untreated DCs from thesame mice (162.3 ± 18.36 and 306.1 ± 24.35 vs. 28.47 ± 5.13 pg/ml, p < 0.05) (Fig. 5B). Furthermore, the level of IL-1b of DCs fromC3H/HeN mice was 6.18 ± 2.00, 56.54 ± 1.59, and 47.08 ± 2.82 pg/ml in the untreated, LPS-treated, and b2GPI-treated groups, respec-tively (Fig. 5C). Interestingly, b2GPI stimulation did not increaseIL-12 production in the DCs from C3H/HeJ mice, but obviouslyincreased the production of TNF-a and IL-1b (p < 0.05).

Fig. 5. Cytokine production by iDCs from C3H/HeN or C3H/HeJ mice afterincubation with LPS or b2GPI iDC (1 � 106) of C3H/HeN or C3H/HeJ mice wereincubated for 12 h with LPS (1 lg/ml) or b2GPI (50 lg/ml). Thereafter, the cellswere collected and centrifuged at 1500 rpm for 5 min, and the concentrations of (A)IL-12, (B) TNF-a, (C) IL-1b were determined in the supernatant by ELISA. The datarepresent mean values ± SEM of three separate experiments. Significant differencesfrom corresponding control are indicated as follows: ⁄p < 0.05; ⁄⁄p < 0.01;⁄⁄⁄p < 0.001; Student’s unpaired t test.

230 J. Liu et al. / Cellular Immunology 290 (2014) 226–232

3.4. The role of b2GPI-treated BMDCs from C3H/HeN or C3H/HeJ micein inducing T cells proliferation

It has been demonstrated that mDCs have an important func-tion to activate T cell responses. In order to detect if b2GPI-treatedBMDCs from C3H/HeN or C3H/HeJ mice have different ability toactivate T cells, DCs after different stimulations were co-culturedwith T cells of BALB/c mice at a DC/TC ratio of 1:20 for 48 h. Anenhanced T cells proliferation could be induced by C3H/HeN

Table 1Proliferation of alloreactive T cells in response to b2GPI-treated DCs.

Lymphocyte proliferation (O.D. at 490 nm)

Without DCs Untreated DCs

C3H/HeN 0.7230 ± 0.058 0.9133 ± 0.007C3H/HeJ 0.7300 ± 0.060 0.9033 ± 0.030

iDCs from C3H/HeN or C3H/HeJ mice were incubated for 12 h with LPS (1 lg/ml) or b2GPcells at a DC: T cell ratio of 1:20 in 96-well plates. T cell proliferation was detected by Cewas monitored by the absorbance at 490 nm using a kinetic microplate reader. The datafrom corresponding control are indicated as follows: ⁄⁄p < 0.01; ⁄⁄⁄p < 0.001; Student’s u

BMDCs after LPS or b2GPI treatment compared with untreatedgroup (p < 0.05). And it was also found by the BMDCs from C3H/HeJ mice after stimulation with b2GPI but not after stimulationwith LPS (Table 1).

4. Discussion

APS is an autoimmune multi-systemic disorder characterizedclinically by recurrent vascular thrombosis, pregnancy morbidity,lupus anticoagulant (LA) and aPL including anticardiolipin anti-body (aCL), anti-b2-glycoprotein I antibody (anti-b2GPI) [11,27].Gladigau et al. [28] reported that endothelial cells and monocytesare activated by aPL in a TLR4-dependent manner. Boonstra et al.[29] reported that TLR4 is expressed at a high level in BMDCs.Moreover, TLR signaling in DCs temporarily enhances antigenendocytosis and autophagy [19]. In the present study, we foundthat TLR4 was involved in b2GPI-stimulated BMDCs maturation.

It has been reported that BMDCs undergo many morphologicalchanges during maturation, such as the formation of veils and den-drites [26]. In order to acquire high motility, filamentous actins aredepolymerized, and vinculin containing adhesive structures arereduced [30]. Compared to C3H/HeJ mice, DCs from C3H/HeN micedisplayed more mature in response to b2GPI (Fig. 1). The resultsuggested that TLR4 may play an important role in the morpholog-ical difference between two types of mice cells.

Next, we explored the expression of DCs surface molecules indifferent mice. CD11c is a type I transmembrane protein that ishighly expressed in DCs [27]. MHCII is an important molecule inDCs and B cells presenting antigen-derived peptides to CD4-posi-tive T lymphocytes [31]. Shin et al. found that the conversion ofimmature to mature dendritic cells results in the redistributionof major histocompatibility complex class II molecules (MHCII)from late endosomal and lysosomal compartments to the plasmamembrane, so the majority of MHCII is retained inside iDCs, whileMHCII in mature cells is mostly expressed at the plasma mem-brane [32,33]. The co-stimulatory molecules, CD80 and CD86, ascrucial secondary signals for the generation of effector T cells arealso related to the maturation of DCs. The capacity of DCs up-regulation of the plasma membrane distribution of MHCII andco-stimulatory molecules may imply the enhancement of activityof DCs. Our results indicated that b2GPI, as a kind of antigen, hasthe similar function as LPS to induce iDCs maturation. We furtherdemonstrated that the number of mature DCs in C3H/HeN micewas significantly increased than that in C3H/HeJ mice under thesame stimulation (Figs. 2–4). The results indicated that TLR4 mayplay a key role in the immune activation of DCs induced by b2GPI.

IL-12 released by DCs is necessary to drive the differentiation ofnaïve T lymphocytes to Th1 cells [17,34]. We further examined theeffects of TLR4 on the secretion of cytokines, such as IL-12, TNF-aand IL-1b. Upon exposure to LPS or b2GPI, DCs from C3H/HeN micesignificantly increased the production of IL-12, TNF-a and IL-1bcompared with untreated group (Fig. 5). Chan et al. reported thatthe production of IL-12 is higher from mDCs than their early pro-liferating precursors, because IL-12 not only increases the number

LPS-treated DCs b2GPI-treated DCs

1.0890 ± 0.071⁄⁄⁄ 1.1770 ± 0.078⁄⁄⁄

0.9867 ± 0.052 1.0600 ± 0.032⁄⁄

I (50 lg/ml). Cells were washed and incubated for 48 h with 2 � 105 alloreactive T-llTiter 96� AQueous One Solution Reagent (40 ll/well) on day 3. Color developmentrepresent mean values ± SEM of three separate experiments. Significant differencesnpaired t test.

J. Liu et al. / Cellular Immunology 290 (2014) 226–232 231

of DCs but also contributes to DC maturation and their function[30,35]. Tumor necrosis factor-alpha (TNF-a) and interleukin-1beta (IL-1b) regulate both local and systemic inflammatoryresponses. They can activate endothelium and then increase thelevels of tissue factor, which can, in turn, lead to local activationof the coagulation cascade. IL-1b stimulates hepatocytes to pro-duce acute phase proteins and amplifies the innate immuneresponse [7]. Interestingly, compared with C3H/HeN mice, b2GPIstimulation failed to increase IL-12 secretion, but obviouslyincreased the secretion of TNF-a and IL-1b in DCs from C3H/HeJmice (Fig. 5). Alard et al. reported that b2GPI on endothelial cellsurface occurs through interaction with TLR2 [3]. Our results sug-gested that TLR4 can regulate the secretion of cytokines to influ-ence the immune response, but it may be not the only receptorof DCs to regulate the secretion of some cytokines upon the stim-ulation of b2GPI in the process of BMDCs maturation.

The proliferation of T cells in a mixed leukocyte reaction (MLR)is commonly used to evaluate the function of DCs [36]. T cells co-cultured with b2GPI- or LPS-treated iDCs from C3H/HeN miceshowed stronger proliferative ability compared to T cells co-cul-tured with untreated DCs. Differently, in C3H/HeJ mice, LPS-treatedBMDCs could not induce T cell proliferation, but b2GPI treatmentshowed some stimulation effects on T cell proliferation by usingMLR assay (Table 1). Previous study demonstrated that TLR2 isrelated to the binding of b2GPI on cells surface [3,37]. In our study,we proposed that there may be other TLRs associated with thefunction of DCs induced by b2GPI [38].

Relevant researches proposed that there are 3 stages in the pro-cess of aPL production: (1) activation of antigen-presenting cells(APCs) and human b2GPI-specific T cells; (2) activation of humanb2GPI-specific B cells; (3) generation of B cell autoimmunity [39].In conclusion, our experiments suggested that TLR4-associatedevents are at least one of the mechanisms involving in the processof mouse BMDCs maturation induced by b2GPI. Whether TLR4 par-ticipating in aPL production involving T cells and B cells reactionsneeds further investigation.

Disclosure

None.

Acknowledgments

This work was supported by National Natural Science Founda-tion of China (No. 81370614) to Hong Zhou, Sci-tech InnovationTeam of Jiangsu Province (No. LJ201116) and Key Laboratory ofCardiovascular disease of Zhenjiang (SS2012002) to Jinchuan Yan,Student’s Scientific Research of Jiangsu University (No. 12A119)to Jingjing Liu, and Graduate Innovation Foundation of JiangsuProvince, China (CXZZ13_0702) to Hongxiang Xie.

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