| ObjectivePreeclampsia is a worldwide leading cause of maternal death and perinatal mortality. Generalized dysfunction of the maternal vascular endothelium is thought to be the central event in pathogenesis of preeclampsia. However, how endothelial cells (ECs) are activated exactly in preeclampsia is still unknown.Recent investigations into preeclampsia have strongly implicated immuno-logic dysfunction as a significant feature of this disease. Studies have shown that intracellular adhesion molecule 1 (ICAM - 1) , vascular adhesion molecular 1 (VCAM - 1 ) , E - selectin and platelet endothelial cell adhesion molecule 1 (PECAM - 1) increased in serum or plasma from women with preeclampsia, and after stimulation with sera from preeclamptic patients, higher amounts of VCAM -1, ICAM - 1, and E - selectin were detected on the endothelial surface. Cell adhesion molecules have been implicated in the activation of immune system, in particular, in the interactions of effecter cells and target cells. The increased expression of adhesion molecules on vascular endothelium enables the adhesion of peripheral blood cells, and may lead to further damage of endothelium.The natural killer (NK) cells are bone marrow derived and comprise 5 -10% of peripheral blood mononuclear cells. NK cells recruit from blood to tissue rapidly following viral and bacterial infections, and are found in the allograft infiltrate during the early stage of rejection. NK cells have increased ability to adhere to and kill ECs upon activation, and play an important role in EC injury associated with lymphokine - activated killer cells therapy and transplantation rejection.All these evidences suggest that NK cell might be one of the possible effectors targeting ECs in preeclampsia. In this study, we demonstrate the involvement of immunological events focusing on the interaction between NK cells and ECs in the pathogenesis of preeclampsia.Methods1. Study populations:Blood samples were collected from 10 severe preeclamptic patients, and 10 normal pregnant women. Each control woman was maternal age and gestational age matched with a woman with preeclampsia. The diagnosis of severe preeclampsia is according to the criteria of the textbook of Obstetrics and Gynecolo-gy 6th edition.2. Preparation of NK cells;Peripheral blood mononuclear cells (PBMC) were isolated from blood of healthy non - pregnant volunteer by Ficoll Hypaque gradient. Red blood cells were lysed by hypotonic shock. In the experiments concerning adhesion assay, NK cells were negatively isolated with saturating amount of mAb antiCD3, an-tiCD14, anti - CD123w, HLA - DR, DQ, and Dynabeads to deplete T cells, monocytes and B cells. Freshly isolated NK cells were suspended in PBS at 107/ ml, and labeled with lOOuCi Cr ( sodium chromate) at 37 C for 3 hours. In the experiments of NK cytotoxicity, functional and morphologic changes of endo-thelium induced by NK in preeclampsia, NK cells were isolated by Percoll.3. Preparation of human umbilical vein endothelial cells (HUVECs) : HUVECs were obtained from umbilical vein by the method of Jaffe et al,and were cultured in medium : 2% fetal calf serum, lOng/ml human epidermal growth factor, 1 |xg/ml hydrocortisone, 50|xg/ml gentamicine, 50ng/ml ampho-tericin B, 5ng/ml human fibroblast growth factor basic( hFGF - B), and 10 (xg/ml heparin. The purity of EC cultures was checked by expression of factor VIII antigen by histochemistry and found to be 99% positive.4. Adhesion assayHUVECs were grown to confluence in flat - bottomed 48 - well plates. 51 Crlabeled NK cells were re - suspended at 5 xlOVml. The labeled cells (0.2ml) was dispensed to each well and incubated for 20 hours at 37%! in 5% CO2 incubator with humidity in presence of 20% sera(0.1 ml serum per well, at final a-mount of 0.5 ml). At the end of incubation, the supernatant was discarded and the wells were carefully washed three times with PBS. The adherent cells were solubilized with 1% Triton X 100. The lysates were collected and counted in a gamma counter. Maximal release is the radioactivity released by the total labeled NK cells which have been dispensed to one well. Spontaneous release is the radioactivity released by the HUVECs in one well. Percent adhesion was calculated as following formula.Adhesion (%) = (experimental release - spontaneous release) / (maximal release - spontaneous release) 100%5. Effect of anti - cell adhesion molecule antibodies on adhesion of NK cells to ECs in severe preeclampsiaHUVECs were grown to confluence in flat - bottomed 48 well plates. HUVECs and NK cells were incubated with 20% sera for 20 hours respectively. HUVEC monolayers were pretreated with anti - ICAM - 1 or anti - VCAM - 1 mAb for 1 h, and NK cells were pretreated with anti - LFA - 1 or anti - VLA -4 mAb for half an hour at 37 "C before co - incubation. Isotype - matched antibodies were used as negative control. 105NK cells were dispersed to each well. At the end of incubation, the supernatant was discarded and the wells were carefully washed three times with PBS. The adherent cells were solubilized with 1 % Triton X. The lysates were collected and counted in a gamma counter. Percent blocking was calculated as following formula.Blocking (% ) = ( Adhesion (% ) of negative control Adhesion (% ) after blocking with mAb) / Adhesion(% ) of negative control 100%6. The effect of severe preeclampti sera on NK killing abilityNK cells were incubated with 20% serum from severe preeclamptic patients and normal pregnant women for 20 hours. The effect of ser pSf on NK killing a-bility to K562cells was analyzed by MTT method. NK killing ability was also analyzed after incubation with 10% and 40% sera from severe preeclamptic patients.7. Aptosis of HUVEC induced by sera -treated NK cells.NK cells were incubated with 20% serum from severe preeclamptic patients and normal pregnant women for 20 hours, and then added into the wells of HUVECs. HUVECs and NK cells were co - incubated for further 20 hours. Aptosis of HUVEC induced by sera treated NK cells was checked by FCM and electronic microscope.8. OSecretion of ET -1 and PGI2 by HUVEC induced by sera - treated NK cellsNK cells were incubated with 20% sera from severe preeclamptic patients and normal pregnant women for 20 hours, and then added into the wells of HUVECs. HUVECs and NK cells were co - incubated for further 24 hours. Levels of ET -1 and PGI2 ( 6 - keto — PGFla ) in the supernatants was checked by ra-dioimmunologic method.Results1. Adhesion of NK cells to HUVECsThe percentages of NK cells adhered to the HUVECs monolayer are shown in Figl. The adhering percentage of NK cells to HUVECs in severe preeclamptic group was 39 ±9% , while 15 ±9% in normal pregnant group0 The adhesion in severe preeclamptic group was increased significantly in comparison with normal pregnant group, P < 0.01.2. Effect of anti - cell adhesion molecule antibodies on adhesion of NK cells to ECs in severe preeclampsia.The adhesion of NK cells to ECs was obviously reduced after each block. The blocking percentages by mAb to LAFA -1,VLA -4,ICAM -1 and IVCAM -1 were 34 12% , 37 27% , 44 23% , and 52 12% respectively in severe preeclampsia .3. The effect of sera on NK killing ability to K562 cellsAfter incubation with 20% sera for 20 hours, NK killing ability to K562 cell was significantly increased in severe preeclamptic group (47.52 ±4.91% ) than that in normal pregnant group (33.32 ±6.53% ). P <0.01.In severe preeclamptic group, the killing ability of NK cells increased significantly with the increasing concentration of sera. P <0. 01. In 10% severe preeclamptic sera group, the killing ability of NK cells was 40. 98 ±5. 68% , while 47.52 ±4.91% in 20% severe preeclamptic sera group, and 57.65 ±8. 14% in 40% severe preeclamptic sera group.4. Apoptosis of HUVEC induced by sera treated NK cells was checked by FCM and electronic microscope.In severe preeclamptic group, AnnexinV positive cells is 24.36 ±4.15% , significantly increased in contrast with normal pregnant group (14. 94 ±4. 01% ), p <0.01;AnnexinV + PI positive cells is 2.94 ±0. 58% , significantly increased in contrast with normal pregnant group (2. 06 ± 0. 31% ) , p < 0. 01;while PI positive cells is 0. 15 ± 0. 07% , no significant difference with normal pregnant group(0.19 ±0.08% ) oUnder EM, typical morphologic changes of apoptosis were shown in severe preeclamptic group.5. Secretion of ET -1 and PGI2 by HUVEC induced by sera treated NK cellsIn the supernatants of NK and HUVEC co - incubation, level of ET - 1 in severe preeclamptic group is 46. 00 ± 11. 47u^/ml, significantly increased in contrast with normal pregnant group(28.73 ±2.98|xg/ml) , p <0.05;level of 6 - keto - PGFlain severe preeclamptic group is 535.41 ±75.69u,g/ml , significantly decreased in contrast with normal pregnant group (686. 01 ±83. 92u,g/ ml), p<0.01.Conclusions1. Sera from severe preeclamptic patients increase the adhesion between NK cells and endothelial cells significantly.2. mAb to LFA - 1, VLA - 4, ICAM - 1 and VCAM - 1 significantly blocked the adhesion between NK cells and endothelial cells in severe pre-eclampsia.3. NK killing ability increased significantly after incubation with 20% serafrom severe preeclamptic patients.4. Severe preeclamptic sera - treated NK cells induce increased apoptosis of ECs5. Secretion of ET -1 by ECs induced by severe preeclamptic sera - treated NK cells increased significantly and that of PCI - 2 decreased significantly.
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