Available online at www.annclinlabsci.org Annals of Clinical & Laboratory Science, vol. 47, no. 4, 2017 389 L- Promotes the Maturation of Dendritic Cells via Up-regulation the Expression of TLR4 in vitro

Zhishuai Ye1, Jia Liu1, Jie Zheng1, Jianing Zhang2, and Rongchong Huang1

1Department of Cardiology, The First Affiliated Hospital of Dalian Medical University and 2College of Life Sciences and Pharmacy, Dalian University of Technology, Dalian City, China

Abstract. The relationship between dendritic cells (DCs) and L-selectin in the progress of atherosclerosis is unclear. Here, we used L-selectin co-cultured with DCs to investigate the effect of L-selectin on the maturation of DCs in vitro. Monocytes derived DCs were isolated and cultured from human peripheral blood. After being stimulated with L-selectin and/or its antagonist for 24-48 hours, the feather of cells was observed by the electron microscope. The expression of mature antigens CD1a, CD80, CD83, and CD86 were investigated by flow cytometric analysisAC (F S). RT-PCR and FACS were used to detect the mRNA and protein expression of Toll-like receptor 4(TLR-4). We found that only the cells of giving L-selectin have the mature special feature for irregular shapes. DCs which were stimulated by L-selectin have a larger number of expressing CD1a, CD80, CD83, and CD86 compared with non-stimulated and cultured with L-selectin antagonist. The transcript levels of TLR4 were significantly higher after L-selectin and lipopoly- saccharide (LPS) stimulated. And the antagonist of L-selectin can deeply decrease the expression of CD1a, CD80, CD83, and CD86 on DCs appeared to coincide with the level of TLR4 transcription. The results demonstrate L-selectin can promote the maturation of DCs via up-regulation the expression of TLR4.

Key words: Atherosclerosis, Dendritic cells, L-selectin, TLR4.

Introduction and immunoglobulin families also in concert with L-selectin molecules to mediate the adhesion and It was first confirmed that atherosclerosis maybe extravasation of leucocytes. have a com- caused by a response to chronic inflammation in mon structure containing an NH2-terminal Ca2+ 1986, and atherosclerosis is an inflammatory auto- dependent lectin, an epidermal growth factor immune disease, chronic inflammation throughout (EGF) to sustain the molecule structure, they also atherosclerosis [1]. contain a variable number consensus repeats of complement regulatory proteins [6]. L-selectin has The key defensive mechanism in the body isthe to combine with ligands in the adhesiveness. There leucocytic extravasation to surrounding tissue. have been confirmed three categories of molecules Leading to adhesion of cells to activated endotheli- as L-selectin ligand: sialomucin, proteoglycans, um is the first step of the immunity cascade by ad- and glycoproteins [7]. A number of known ligands hesion molecule. The selectin family is the most like the mucosal ones address in-cell adhesion important of adhesion molecules in the early period molecule-1(MadCAM-1), glycosylation-depen- of response, and it consists of the three closely ho- dent -1(GlyCAM-1), mologous glycoproteins such as E-selectin, P-selectin glycoprotein ligand-1 (PSGL-1) and P-selectin, and L-selectin. E-selectin is expressed on sLex [8,9]. Shedding of L-selectin appears to be endothelial cells, while P-selectin is expressed in regulated by leucocytes after activation. However, both endothelial cells and platelets. L-selectin, on the physiological significance and mechanisms of the other hand, is constitutively expressed on the shedding L-selectins are still not completely un- majority of leukocytes [2]. L-selectin exhibits adhe- derstood. L-selectin plays an important role in sive as well as signaling functions [3,4] and is par- both acute and chronic inflammatory response. ticularly important for lymphocyte homing to sec- Recent studies show that L-selectin in patients ondary lymphoid organs [5]. Members of integrin with coronary artery disease has a higher level as compared to healthy people, and the level of Address correspondence to Rongchong Huang, MD; Professor of Medicine/Cardiology, The First Affiliated Hospital of Dalian Medical L-selectin is related to the stabilization of University, Dalian 116011, China; e mail. [email protected] atherosclerosis.

0091-7370/17/0400-389. © 2017 by the Association of Clinical Scientists, Inc. 390 Annals of Clinical & Laboratory Science, vol. 47, no. 4, 2017

Figure 1. Analysis of different L-selectin concentration for DCs maturation. (A) Flow cytometry data of CD1a, CD80, CD83 and CD86 of DCs after co-culture with different L-selectin concentration from 75ng/ml to 200ng/ml. (B) Statistical analysis results from four independent groups respectively. Data are expressed as Mean±SD;*p<0.05 100ng/ml L-selectin vs. 75ng/ml L-selectin; #p<0.05 100ng/ml L-selectin vs. 200ng/ml L-selectin.

Dendritic cells (DCs) are specialized antigen pre- Materials and Methods senting cells (APCs) which have unique ability to induce a primary immune response by activation of Generation of dendritic cells from monocytes. This in- naive T cells [10]. Their immune function depends vestigation was performed with approval by the Ethics on their maturation. In most tissues, DCs are pres- Committee of the First Affiliated Hospital of Dalian Medical University, China. Informed consent was ob- ent in an immature state and are unable to stimu- tained from blood donors. The principal method for late naive T cells. Mature DCs have the special tree- generating BMDCs was adapted from that published by like shapes and they obtain the ability of Parlato et al. with minor modifications [14]. Briefly, two downregulation of endocytotic activity, but upreg- hundred milliliter blood was obtained from each donor. ulation of adhesion molecules (CD11a, CD50, The mixture of containing white cells and lymphocyte CD54 and CD58), co-stimulatory molecules separation medium in centrifuge tubes were density gra- (CD40, CD80/B7.1, CD86/B7.2) and antigen dient centrifuged at 2000rpm for 30 min using presenting molecules [10-12]. However, the role of Histoplaque 1077 (Sigma, St Louis, MO, USA). The DCs in atherosclerosis-related autoimmunity has pure monocytes were collected anti-CD14+ microbeads not been clearly studied. The presence of dendritic (Miltenyi Biotec, Auburn, CA, USA) and seeded into 6-well plates, cultured in 3ml RPMI 1640 medium cells in the aorta and atherosclerotic lesions sug- (Gibco, USA) containing 5% fetal calf serum (Gibco, gests that presentation of lesion antigens is impor- USA), 100ng/ml recombinant human granulocyte-mac- tant in atherosclerosis development [13]. rophage-colony-stimulating factor (rhGM-CSF, Peprotech, NJ, USA) and 50ng/ml recombinant human Both dendritic cells (DCs) and L-selectin are relat- interleukin-4 (rhIL-4, Peprotech, NJ, USA). On day 5, ed to atherosclerosis. But it is unclear that whether cells were treated with phosphate buffered sodium (PBS) the interaction of L-selectin on DCs is involved in or lipopolysaccharide (LPS)(1ng/mL, Sigma, St Louis, the progression of atherosclerosis. The purpose of MO, USA) or L-selectin (75-200ng/mL, ProSpec, USA) this present study is to observe the effect on DCs or L-selectin antagonist Fucoidan (5g/ml) ((Sigma, St differentiation and immune function by different Louis, MO, USA) for 48 hours. Our previous study has shown that LPS (1ng/ml) could effectively induce DC concentration of L-selectin. maturation and did not significantly promote apoptosis L-selectin induce maturation of DCs by TLR4 391

Figure 2. L-selectin promotes immature DCs (A,×6000) to mature DCs (B,×8000), which was observed by transmission elec- tron microscope. and this concentration of LPS was therefore used in this Flow cytometric analysis of protein expression of TLR-4. study. Until now, there is no related literature about the Cells (5×105) were harvested and blocked with 10% normal proper concentration of L-selectin incubated with DCs. goat serum for 15 minutes at 4°C, washed, and then stained So we refer to the concentration of other selectins and with PE-conjugated mAbs against TLR-4 ( eBioscience, choose the gradient of 75 ng/ml, 100 ng/ml and 200 ng/ USA) for 60 min at 4°C. After immunofluorescence staining, ml. According to the level of mature DCs stimulate by cells were analyzed by FACS Calibur using Cell Quest soft- L-selectin, we could confirm the proper concentration of ware (Becton Dickinson). L-selectin. The cell viability of each group was measured using Tr ypan blue exclusion method and cell viability Statistical analysis. All experiments were repeated at least 3 was over 90% in all groups. times with different cells. Data were expressed as the mean ± standard deviation (SD). Statistical significance among the Flow cytometric analysis for DCs maturation mea- experimental groups was examined by one-way analysis of surement. Cells (5×105) were harvested and blocked variance (ANOVA), followed by the Student-Newman- with 10% normal goat serum for 15 minutes at 4°C, Keuls post hoc test for statistical significance. A p value less washed, and then stained with PE-conjugated mAbs than 0.05 was considered statistically significant. against CD1a, CD80, CD83 and CD86 (all from Becton Dickinson, San Diego, CA, USA) for 30 minutes Results at 4°C. Control immunostaining was performed using the respective nonimmune IgG; no specific staining was The proper concentration of L-selectin for stimulat- observed. After immunofluorescence staining, cells were ing DCs. The L-selectin concentration gradient of analyzed by fluorescence-activated cell sortingAC (F S) 75ng/ml, 100ng/ml and 200ng/ml was chosen for Calibur using Cell Quest software (Becton Dickinson). stimulating DCs. The flow cytometry data of CD1a, RNA isolation and reverse transcription-polymerase CD80, CD83, and CD86 showed that the L-selectin chain reaction (RT-PCR). Total RNA was extracted concentration of 100ug/ml can make the highest level from DCs using a Protein & RNA Extraction Kit of DCs expressing reference proteins (Figure 1). After (Takara, Bio Inc., Shiga, Japan) without phenol and gua- giving L-selectin of 200ng/ml, the state of DCs was nidine isothiocyanate. Selected primers were: worse and the expressed antigen of DCs was down-reg- GAPDH (F) 5′-ACCACAGTCCATGCCATCAC-3′, ulated significantly. So the proper concentration of (R) 5′-TCCACCACCCTGTTGCTGTA) -3′; L-selection which can promote DCs to mature period is TLR-4 (F) 5′-ATTCCGATTAGCATACTTAG-3′, 100ng/ml. (R) 5′-TGGAGGGAGTTCAGACAC-3′. RT-PCR was performed with a Prime Script® II High Fidelity RT- PCR Kit (Takara, Bio Inc., Shiga, Japan) under the fol- L-selectin promotes DCs maturation. As shown in lowing conditions: denaturation at 95ºC for 5min, 30 Figure 2, DCs with L-selectin have mature special fea- amplification cycles (98ºC for 10 sec, 55ºC for 15 sec, ture for irregular shapes in many dendritic cells. The and 68ºC for 1 min), and extension for 5 min at 72ºC. L-selectin co-exist with DCs have typical numerous cy- The amplified products were separated by agarose gel toplasmic processes, the surface of cells is rough, abun- electrophoresis on a 2% gel and analyzed by BioImaging dant of cytoplasm and the organelles in the cytoplasm Systems (UVP, Labworks, ver. 4.6). less than the immature group. 392 Annals of Clinical & Laboratory Science, vol. 47, no. 4, 2017

Figure 3. L-selectin antagonist Fucoidan decreases DCs maturation. (A) The flow cytometry data of CD1a, CD80, CD83 and CD86 of DCs after untreated, co-culture with LPS, L-selectin (100ng/ml) and L-selectin plus Fucoidan. (B) Statistical analysis results from four independent groups respectively. Mean±SD;*p<0.05 100ng/ml L-selectin vs. control group; ^p<0.05 100ng/ml L-selectin vs. LPS; #p<0.05 100ng/ml L-selectin vs. L-selectin+Fucoidan.

L-selectin’s antagonist decreases DCs matura- Discussion tion. It was found that L-selectin stimulated DCs expressing a high level of CD1a, CD80, CD83, In the present study, we found for the first time that and CD86 compared with non-stimulated and L-selectin could induce DCs maturation, which L-selectin antagonist groups. The groups of giving had morphological characteristics of typical mature both L-selectin and its antagonist can decrease the DCs as well as up-regulated the expression of co- level of expressing CD1a, CD80, CD83 and CD86 stimulatory molecules (CD80, CD86) and relative (Figure 3). marker proteins (CD1a, CD83). L-selectin was L-selectin up-regulates expression of TLR4 in found to promote the maturation of DCs via TLR DCs. The result of RT-PCR showed that transcript signal transduction pathway. It was thus revealed levels of TLR4 were significantly higher in the that DCs were, in the presence of L-selectin, in- L-selectin groups and the LPS groups than in non- volved in the rolling and adhesion with vascular stimulation and in L-selectin antagonist. And the endothelial cells, and became mature DCs by anti- antagonist of L-selectin can deeply decrease the gen absorption and processing. DCs then activated level of TLR4 transcription. Similar results could T cells and triggered an inflammatory response by be collected for the proteins of TLR4 expressing on presenting internal or external antigens. DCs. Whereas in L-selectin antagonist and in non- stimulated, there only few DCs expression for Dendritic cells (DCs) constitute a family of special- TLR4. The frequencies of TLR4 expression on ized antigen-presenting cells, which play an impor- DCs were significantly higher in L-selectin and in tant role in the occurrence and development of AS LPS stimulated groups. Compared with the group [13,15]. But the mechanism of DCs triggering and of giving L-selectin, the specific of L-selectin an- amplifying immune and inflammatory response is tagonist can significantly downregulate the protein still unknown. In the immune and inflammatory expression of TLR4 (Figure 4). response, the effusion of leucocytes is a key step in L-selectin induce maturation of DCs by TLR4 393

Figure 4. L-selectin up-regulates DCs mRNA and protein expression of TLR4. (A) The flow cytometry data of TLR4 protein of DCs after untreated, co-culture with LPS, L-selectin (100ng/ml) and L-selectin plus Fucoidan. (B) RT-PCR shows the TLR4 mRNA of DCs after untreated, co-culture with LPS, L-selectin (100ng/ml) and L-selectin plus Fucoidan. Mean±SD;*p<0.05 100ng/ml L-selectin vs. control group; ^p<0.05 100ng/ml L-selectin vs. LPS; #p<0.05 100ng/ml L-selectin vs. L-selectin+Fucoidan. triggering the defense mechanism of the organism. inhibited when L-selectin concentration reached L-selectin, as an important member of adhesion 200ng/ml: cell debris increased and cell permeabil- molecules, participates in the adhesion of leuco- ity decreased, probably due to a high concentration cytes to endothelial cells in the early stage of the that interfered with the normal activities of the inflammatory response. It has been found that the cells. We found with flow cytometric detection that migration of immature DCs to inflammatory tis- L-selectin of 100ng/ml could upregulate the ex- sues depends mainly on the mediation by selectin pression of the four antigens at the surface of DCs. family of adhesion molecules [16]. Therefore, the A significant increase was witnessed compared to present experiment chose L-selectin as interference the immature DCs with specific phenotypes of ma- factor, to observe its influence on DCs morphology ture DCs. Fucoidan could partly inhibit the posi- and phenotypes in vitro and the mechanism of its tive rate of the four antigens expressed by DCs. influence. The antagonist of L-selectin has nospe- Therefore, 100ng/ml was determined to be the op- cific antibody so far. It has been reported Fucoidan timum interference concentration of L-selectin. is a sulfated polysaccharide extracted from brown The identification, engulf and presentation of anti- seaweeds that possess anti-inflammatory effects gens are mediated by the pattern recognition recep- [17]. Fucoidan has been shown to act as a ligand for tors (PRRs) on the surface of the DCs. PRRs con- either L- or P-selectin, thereby inhibiting leukocyte sist of TLRS, C-type lectin, RLRs, etc. TLR is the rolling [18,19].Therefore, Fucoidan was chosen as only one that directly mediates the maturation of the inhibitor to observe its influence on the matu- DCs [20]. In atherosclerotic plaques are found the ration of DCs. expression of large quantities of receptors TLR2 and TLR4, which are mainly distributed in im- But up to now, there has been no reports specifying mune cells such as DCs and . Studies the optimum concentration of L-selectin for its in- have demonstrated that early-stage AS can activate terference on DCs. Therefore, we referred to the inflammation, such as the chemotaxis, migration, optimum concentration of relevant selectins, and and adhesion of DCs and macrophages by TLR4 used the concentration gradient method. 75ng/ml, signal pathway [21]. TLR4 promotes the matura- 100ng/ml and 200ng/ml were respectively chosen. tion of DCs and mediates the inflammatory re- Flow cytometric detection showed that L-selectin sponse by antigen processing, up-regulating co- reached its optimal state at the concentration of stimulatory molecules and cell factors. MHC-I, II 100ng/ml, which could cause DCs to express the will have significantly increased expression on the four antigens CD1a, CD80, CD83 and CD86 at a surface of DCs stimulated by antigens. Its capacity high level. Cell activities were significantly of charging MHC-I and MHC-II will increase as 394 Annals of Clinical & Laboratory Science, vol. 47, no. 4, 2017

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