Study Of The Change Of Immune Molecules On HUVEC By LPS Stimulation And It's Mechanism

BackgroundVascular endothelial cells(VEC) are unique multifunctional cells with critical basal and inducible metabolic and synthetic and immune functions. They form the lining of the circulatory system , constitute the interface between the blood and the tissue . As a selective permeability barrier, it not only modulate blood vessel tone and thrombosis, but also regulate inflammatory response through secreting pre-inflammatory factors and controlling leukocytes interaction with vessel wall. The structure and function will change if it is stimulated by endogenous and exogenous stimuli. LPS is an important factor which can directly and indirectly activate VEC. In recent studies, the direct receptor-mediated activation signal is the focus, now lots of LPS receptors have been found and identified. Among these, the study about the TLR4 has been the hot and has got many results.After stimulated by LPS, endothelial cells shrink immediately, express P-selectin, Von. Willerbrand factor and then secrete cytokines , express adhesion molecules, recruit the leukocytes, mediate the inflammatory response. As non-professional APCs, ECs have its own characteristic in the expression of immune molecules on un-activated and activated endothelial cells. Recently, more researches have indicated that activated endothelial cells could express MHC-â…  and MHC-â…¡ molecules,CD40,CD58,ICOS-L,OX40-L costimulating molecules, which take part in and regulate adaptive immune response .Today, to identify and study the function of immune membrane molecules on Ecs become a very hot and key issue.Objective1. To compare the change of immune molecules and cytokine between the un-actived and activated endothelial cells by LPS stimulation.2. To explore the possible mechanism by which actived HUVEC influence immuneresponse.Methods To achieve our goals, we conducted the following works:1. Primary culture of HUVEC: HUVEC were obtained from healthy mature infant umbilical cord by separated and incubated. HUVEC were identified by morphologic character and membrane antigen Vffl factor.2. Observation of the production of cytokine from HUVEC : IL-6, IL-8> TNF- a in supernatants were measured by ELISA at 24h following 0,50,100, lOOOng/ml LPS stimulation.3. Detection of the expression of membrane molecules : Total RNA was extracted from HUVEC with and without LPS stimulation, TLR4,B7-HKB7-H2,B7-H3.B7-H4,B7-DC> LIGHT, HVEMs TR6, LTPR, HLA-DR, CD80. CD86, OX40L> OX40mRNAwere detected by RT-PCR and analysed by Bandscan software.4. Observation of the expression of CD40. ICAM-K CD137 : HUVEC with and without LPS stimulation were collected ,and analysed the expression of CD40, ICAM-1, CD137byFACS.5. Detection of the production of cytokine from HUVEC by LPS after blocked by anti-TLR4mAb.: Blocking the interaction between LPS and TLR4 with anti-TLR4 mAb , the production of IL-6n IL-8> TNF- a were measured with ELISA.6. Observation of the expression of membrane molecules by LPS after blocked by anti-TLR4mAb: Blocking the interaction between LPS and TLR4 with anti-TLR4mAb, establishing the standard curve with ï¿¡ -actin, membrane molecules B7-HK B7-H2> B7-H3> B7-H4, B7-DC, HVEM> TR6, LT P R, CD40, CD137 were deteced by real-time PCR.7. Statistical analysis for the data was worked-out with SPSS 10.0. Results1. Primary culture of HUVEC was successful with positive staining with the Von. Willerbrand factor.2. HUVEC without LPS stimulation could express TLR4> B7-H2, B7-H3. TR6. LT0R> B7-DC>HVEM mRNA, except for B7-DC>HVEM ,the expression levels were up-regulated by LPS. The expression of OX40L, B7-H1, B7-H4mRNA were inducible.CD80, CD86, HLA-DR, LIGHT, OX40 mRNA were not detectable both before and after LPS stimulation.3. HUVEC without LPS stimulation could slightly express CD4(h ICAM-1 ^ CD 137 with FACS, and the expression levels were obviously up-regulated by LPS.4. HUVEC without LPS could secrete a small quantity of IL-6> IL-8, TNF-aandthe production was obviously enhanced by LPS stimulation in dose-dependent .But the production of TNF- a is lower than the production of IL-6and IL-8.5. After blocking the interaction between LPS and TLR4 with anti-TLR4mAb, the production of IL-6> IL-8> TNF-a were obviously decreased; the expression of B7-H2> B7-H3, TR6> LT P R> CD137> CD40 which influenced by LPS and B7-H1, B7-H4 which was inducible were inhibited in different level. The change of the expression of B7-DC and HVEM was not detected .Conclusion1. The result of RT-PCR> real-time PCR and FACS, for the first time, showed that B7-H3> B7-DC> CD 137 were constitutively expressed on HUVEC and B7-H4 was induciblely expressed.2. By detecting immune membrane molecules on HUVEC with and without LPS stimulation, HUVEC, as non-professional APCs , could not activate native T cells owing to the lack of CD80 and CD86, but could activate and regulate memery T cell and actived T cell through the constimulating signal from B7 family (including B7-HK B7-H2> B7-H3^ B7-H4n B7-DC)> CD40and OX40L and the assistance of LIGHT and its three receptors, negatively regulated the immune response by these negative modulatory signals from B7-HK B7-DC> B7-H3> B7-H4atlast.3. The result which the production of IL-6^ IL^ TNF-a was enhanced by LPS stimulation showed that HUVEC could form the cytokines regulated network through recruiting leukocyte and promoting T cell proliferation, and then mediated inflammation and modulated immune response.4. The production of IL-6, IL-8 and TNF-a was significantly inhibited after blocked by anti-TLR4mAb, indicating that the secretion of cytokine was related to TLR4 signaling pathway. And the expression of membrane molecules up-regulated by LPS also was slightly down-regulated in different level, suggesting maybe have other receptor and signaling pathway besides TLR4.