The Analysis Of The Development And Cell Cycle Of Murine Bone Marrow-derived Dendritic Cells With LPS Stimulation

Dendritic cells (DCs) are one of the most potent antigen presenting cells (APCs). They are distributed widely throughout the body, where they are variously represented by cells such as interdigitating reticular cells (lymphoid organs), blood monocytes, and Langerhans cells (epidermis). They are heterogenous cells that display multiple differences in morphology, phenotype and function. In general, immature DCs, which have a great proclivity for endocytosis, migrate via the vasculature from the bone marrow into various organs, where they reside in an inactive state. Immature DCs do not effectively induce primary immune responses because they express neither the requisite costimulatory molecules nor stable complexes of antigenic peptides with major histocompatibility complex (MHC) molecules. In the presence of inflammatory signals they rapidly take up foreign antigens and undergo a differentiation/maturation process that downregulates further antigen-processing capacity, but enhances their expression of MHC, costimulatory, and other molecules important for successful antigen presentation. They then migrate to the lymphoid organs where they interact with, and activate, naive T cells.The DC system comprises a large collection of subpopulations with different functions. Both phenotypic and functional criteria have allowed the classification of mouse DCs into six main subpopulations: CD8~-DCs, CD8~+DCs, CD8~intDCs, Langerhans cells, Dermal DCs and B220+DCs. Each has different functions. For example, CD8~-DCs induced a vigorous proliferative response in CD4~+T cells, whereas CD8+DCs induced a lesser response that was associated with Fas-dependent T cell apoptosis in vitro. It has been proposed that plasmacytoid B220+DCs are involved in the maintenance of T-cell tolerance by inducing the differentiation of T regulatory cells. It has also been established that immature DCs were thought to induce T cell anergy or regulatory T cells, whereas the same cells, transformed after stimulation into immunogenic mature DCs, represented the unique inducers of effector T cells.Because of the critical roles DCs have in the generation of primary immune responses, an important avenue of investigation is their potential for modulating functions such as the induction of immune tolerance or tumor immunity.The maturational processes of DCs are efficiently regulated, such that these cells can achieve different states of activation/maturation and thereby different functionalproperties, depending on the precise nature of the signals they receive from their microenvironment. Maturation can be triggered by multiple stimuli, including contact allergens, bacteria and viruses, proinflammatory cytokines such as interleukin (IL)-1, IL-6, and tumor necrosis factor-alpha (TNF-a), immunostimulatory unmethylated CpG oligonucleotides, poly (I:C), and signaling molecules (CD40 ligand). Lipopolysaccharide (LPS), the gram-negative microbial cell-wall component, is also a potent inducer of DC (and B cell and monocyte) maturation that facilitates the production of inflammatory cytokines and immunologically important cell-surface molecules by these cells and enhances their antigen presentation.A technology that identifies differential gene expression provides an important tool to cell biologists, and several such methods have been available (e.g., Northern blotting, RT-PCR, differential display, and RNAse protection assays). However, a new method, DNA array analysis that can simultaneously probe hundreds of genes in a sample has recently been developed. The advantage of these DNA arrays is that the genes to be assessed are meticulously selected for their biological relevance. Microarray approach allows quantitative and simultaneous analysis of gene expression of a large amount of genes. Many cellular processes are regulated by changes in mRNA levels. Thus systematic studies of gene expression patterns have proven to be extremely useful for studying cellular effects of natural stimuli and to be a powerful tool to identify molecular events and key pathways involved in specific cellular functions.It is well known that LPS has a lot of affect to dendritic cells. However, the molecular biology of the differentiation and maturation of DCs, especially the affect of LPS to the development and cell cycle of DC based on a global analysis of gene expression, has been less studied. The gene expression profiles of human monocyte-derived DC, as determined by serial analysis of gene expression (SAGE) or DNA array, have been documented. However, similar studies that target mouse bone marrow (BM)-derived DCs have not yet been reported. In the present study, we systematically analyzed the gene expression profiles of mouse BM-derived DCs by DNA array approach.Conventional method was employed to culture murine bone marrow derived dendritic cells (BMDCs) induced by GM-CSF. Among bone marrow cells the CD34+ precursors will differentiate into immature (BMDCs) induced by GM-CSF. In the process the group with sustained LPS stimulation was aimed to study the effect of early administration of LPS on the development of DCs, termed early DCs(eDCs).Conversely, delayed administration of LPS in the last two days(from day 5 to day 7)was called later DCs(lDCs). The group without LPS was used as negative control.After seven days of culturing, DCs were harvested to perform the experiments listedas follows: CFSE staining, MTT, DC synchronization, microarray and affymetrixGenechipPart one: Observe the development of DC by using CFSE staining and MTT approach1. MTT was used to exam the number of DCs from day 3 to day 10.In the control group we can see that the OD value increase which means the increase of DC cell numbers from day 3 to day 6, when was the top, after a steady state for 2 days, the OD value drops gradually; however, the OD value of eDCs increased slightly during day 3 to day 7, then we saw a continues drop. The effect of LPS to lDCs is similar with the eDCs at early stage (up to day 4), when we add LPS. But from then, reverse the tide.2. CFSE staining was used to examing the proliferation of dendritic cells.CFSE is membrane permeant and can diffusion into the intracellular environment. Endogenous esterases render the molecule highly fluorescent. CFSE reacts with free amine groups of intracellular proteins, forming dye - protein adducts and is partitioned with remarkable fidelity between daughter cells allowing eight to 10 discrete generations to be identified both in vitro and in vivo. The division cycle number is analysed by flow cytometry. In our study, we saw gradual CFSE decrease of control group which means DCs proliferation from day 3 to day 10 of culture; however, as for the eDC and the lDCs, we did not see notable division. This indicates that both sustained LPS culture and once LPS stimulation can inhibit DC from proliferation, which is in accordance with the result of MTT and our array data. 3. synchronization of DCIn our study, we had tried to synchronize DCs, which can facilitate our study of the cell cycle of DCs. However, to our knowledge, there are no literatures about synchronization of DC. Therefore, we can only refer to literatures about synchronization of other types of cells. Thymidine, Methotrexate, L-Mimosine, Nocodazole, Hydroxyurea, Aphidicolin and some other kinds of reagents which can synchronize other types of cells were used soly or combined, but we can not make it. The result showed that Nocodazole and Hydroxyurea could synchronize DCs partly,but not completely.Part two: Analyzing the difference of gene expression profilings between the three group by DNA array.As is well known, LPS can serve as a stimulator to make DC mature and active. Our study is aiming at whether LPS can have the similar affect when LPS is administrated during the first stage of culture. To find out this, we used Atlas mouse cDNA express array of CLONTECH Corporation and its software to analyses the difference of gene expression profilings. As the gene number of this array is limited, then Affymetrix mouse gene chip (cat# 430-2A ) was used to further our study.As the result shown, the number of the changed gene of the three groups (eDC vs IDC, the eDC vs Control and the IDC vs Control) is 3025(13.3%) > 939(4.\%), 2068(9.1%) ,respectively, which means a obvious difference. Therefore we used GCOS software from affymetrix company and some other bioinformatics methods to further analysis the gene chip results. Because so many genes expressed in each group, we classified genes into several groups:? genes related with or regulating cell growth: such as cell cycle protein(cyclins), CDK(cyclin dependant kinases) and some other genes correlation withapoptosis.(2) member of cell surface protein including CDs, MHC member, cell adhensive moleculars, receptors, especially the chemokins and their receptors(3) cytokines interleukins, colony stimulates the factor (CSF), interferon (IFN), the tumor necrosis factor (TNF), and some unclassificationed secreted protein.(4) moleculars related to cell signaling transductive passways(5) genes related to antigen processing and presentations, metabolic enzymes and other enzymes such as kinases.All of these genes have covered nearly every aspect of the DC biology. Some of them are in line with our laboratory and other researchers, and some not. Together, our data suggest that DCs experienced a great biological change in sustained LPS stimulation and this maybe a reason for some diseases such as SIRS (systemic inflammatory reaction syndrome) which have a high concentration of LPS in the patient's blood.So, our data have a profound implication to this kind of diseases.Taken together, in our study, we studied different regulating function of the inflammatory factor LPS on dendritic cells' growth, proliferation, differentiation and maturation, especially the difference between the sustained LPS-stimulated DC, onceli...