Preliminary Research Of The Mechanism Of Apoptosis In U937 Cells By Mannan-binding Lectin

As a key molecule in innate immunity, the connections of MBL with infection and immune deficiency diseases have always been a hot subject for research. Some reports show that MBL could regulate the differentiation and maturation of immune cells and the phagocytosis of macrophages Studies suggest that MBL may induce the apoptosis of HeLa cells through binding a certain mannose-containing receptor on HeLa cell surface thereby initiating downstream caspase cascade. It has been shown that surfactant proteins A and D (SP-A and SP-D), which is belong to the collectin subgroup of the C-type lectin superfamily along with MBL together, have similar role in innate defense from infections and various microbes. SP-A and SP-D can bind to various apoptotic cells and enhance engulf of them by phagocytes in Ca2+ -dependent/independent manner. C1q, a key component of the classical complement pathway has a similar structure with MBL. Both C1q and MBL can recognition of the apoptotic cell surface and enhance the uptake of apoptotic cells into a variety of immune cells, such as MΦand imDCs. For example, calreticulin (also known as the C1qR) is bind to CD91 can enhanced engulfment of whole apoptotic cells, as well as cells debris. MBL can bind fragmented DNA and RNA from bacteria and apoptotic cells in Ca2+-dependent manner and facilitate the phagocytosis of them by phagocytes. It can be seen that many members of collectin family play an important role in apoptosis and clearance of apoptotic cells. We have found that MBL inhibited the proliferation of Jurkat cells.Currently, the studies of MBL on the role of apoptosis is few.In this study, MBL was purified from freshly frozen human plasma so that it was of high biological activity and free of LPS. We selected human monocyte line U937 cells as our cell model. We investigated the binding of MBL with U937 cells, the effect of MBL on their growth and the mechanisms of the functions of MBL in induced cells apoptosis.PartⅠExtraction and purification of MBL from human plasma and then removal of LPSThe concentration of MBL in human plasma is quite low, it is between 40～3500μg/ml in normal persons and approximately 900μg/ml in average. The purification of MBL relies on its Ca2+ -dependent affinity for mannan. The experiment adopted affinity chromatography on mannan-Sepharose 4B to purify MBL, which was constructed by Ph.D. Cheng Y and Ph.D. Wang MY from our lab, two steps of affinity chromatography on mannan-Sepharose 4B of pretreatment human plasma, and eluated with D-mannose, EDTA.Popolysaccharide (LPS) is the component of cell-wall of Gram-negative bacteria..It has been reported that LPS plays a vital role in differentiation and maturation of Mo/MΦin many literatures. As we know, U937 cell are human monocyte line, the removal of LPS was the basis of the reliability of the experiment. Therefore, we removed LPS and MBL that bound LPS through Endotoxin removing gel and then use Limulus Amebocyte Lysate (LAL) assay to test the effect of removal. The results demonstrated a significant difference in the concentration of LPS before and after LPS removal. Thus, passing protein column L can effectively remove LPS. In order to prove MBL is accord with the experiment require, we test the biology activity and immunology activity of purified MBL:SDS-PAGE and Western blot show that the purified MBL is compose of high degree of polymerization MBL,which the molecular weight is> 200 000 Da. Ligand binding assay indicated that purified MBL had immunological activity while mannan binding experiments showed that purified MBL maintained biological activity. All of these guaranteed the reliability for subsequent experiments.PartⅡThe effect of MBL on the proliferation and apoptosis of U937cellsIn recent years, studies have found that mannose-binding lectin facilitate the apoptosis of Hela cells. SP-A, SP-D, C1q and MBL play a facilitating role in the phagocytosis of apoptotic cells through immune adjustment. In the mean time, we found inhibited proliferation of U937 cells when provided with high concentration of MBL in its culture. Given that members of collagen lectin family were homologous in terms of structure and function, we inferred that MBL was likely to inhibit cell proliferation by inducing apoptosis.Firstly, we conducted ELISA confirm that MBL could bind U937 cells, thus guaranteeing the reliability for subsequent experiments. Added CCK-8 to cultured cells with different concentrations of MBL after 72h and tested A450nm value after placing it in 37℃incubator for two hours. Applied the same method to cells and then stained cell nuclei with Hochest 33258 to observe the integrity of cell nuclei. Quantified early, late apoptosis rate by using Annexin V-FICT/PI flow cytometry double staining method (time and dose). Determined it was apoptosis or necrosis that caused cell death by adopting DNA ladder electrophoresis.CCK-8 results indicate that the proliferation of U937 cell was significantly inhibited when MBL contribution reached 30～50μg/ml. Hoechst33258 and AnnexinⅤ/PI results showed not only inhibited cell proliferation but also damaged cell membrane integrity, which suggested apoptosis. At first, the nuclei were evenly stained. However, as MBL concentration and application time increased, uneven dyeing chromatin and nuclear fragmentation gradually emerged and cell apoptosis rate rose accordingly. According to light microscopic examination of cells that had been cultured for 54 hours, cells from control groups proliferated significantly and even formed clusters while those from MBL50μg/ml group were sharply below the average in terms of volume and performance. Fragmentation of chromosome indicated by DNA ladder electrophoresis confirmed that it was apoptosis rather than necrosis that caused cell death. In short, applying 30μg/ml～50μg/ml MBL to U937 cells for 72 hours can promote apoptosis in a dose-dependent approach. However, the mechanism requires further investigation.PartⅢInvestigation on the mechanism of MBL in promoting the apoptosis of U937 cellsApoptosis is a gene-controlled programmed cell death and subject to the synthesis of new RNA and protein. There are three pathways to apoptosis:death receptor pathway, the mitochondrial pathway and the CTL-mediated granzyme pathway. Death receptor pathway of apoptosis can recruit FADD through the binding of death receptors (Fas, TNFR1, etc.) and the corresponding ligand (FasL, TNF, etc.). The mitochondrial pathway of apoptosis can regulate mitochondrial membrane permeability with cytochrome c through anti-apoptotic members (Bcl-2, Bcl-XL) and pro-apoptotic members (Bax, Bid). Both pathways ultimately lead to the activation of caspase which catalyses intracellular substrates cleaved as well as cell structure and metabolism destruction, causing biochemical and morphological changes in apoptosis. We attempted to analyze the expression of genes and proteins that might participated in apoptosis through PT-PCR and WB with the goal of discovering the pathways and mechanism of apoptosis. After applying MBL of different concentrations to U937 cells for two hours, we disintegrated cells to harvest proteins or RNA and analyze the expression of proteins and genes from different groups by using Western Blot and RT-PCR. Results showed that the expression of gene Fas and Caspase3 increased, that of gene Bcl-2 and Bax remained unchanged, the expression of protein Fas and FasL rose and apoptosis executor Caspase-3 and protein PARP that partook in DNA damage repair were activated. All demonstrated that MBL could activate death receptor pathway through up-regulation of Fas receptor on the surface of U937 cells and cleave intracellular substrate in a Caspase-dependent manner to induce apoptosis which lead to inhibited U937cellS proliferation.