Available online at www.annclinlabsci.org Annals of Clinical & Laboratory Science, vol. 47, no. 4, 2017 389 L-selectin 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. Selectins 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 cell adhesion molecule-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).