Increased T-cell Chemotaxis Responsed To Staphylococcus Enterotoxin B Induced Endothelial Cell Damage

OBJECTIVE:The incidence of sepsis from Staphylococcus aureus has progressively increased in the last two decades, more important, the mortality from Staphylococcus aureus sepsis shock remains as high as 55.6% despite of enough initial resuscitation, early and aggressive antibiotic use, stress ulcer prophylaxis, and improved nutritional and fluid support. There is increasing experimental evidence that fundamental differences exist in the host response to Gram-positive bacterial pathogens compared with the host response to Gram-negative organisms, which necessitates reevaluation of many of the basic assumptions about the molecular pathogenesis of septic shock. Accumulated evidence was directed towards relationship between staphylococcal enterotoxin as superantigens and pathogenesis of septic shock. In clinical studies, however, evidence for an involvement of superantigen in sepsis has been difficultly to obtain. A important reason is the lack of tools to analyze superantigen effects in patients.Therefore, this study was undertaken to clarify the relation of superantigen with Staphylococcus aureus sepsis by investigating the change of T-cell chemotaxis responsed to Staphylococcus enterotoxin B and it's potential for inducing endothelial cell damage.We carefully observed expression of chemokine receptor on T cell, secretion of cytokine and proliferation simultaneously after purified peripheral blood mononuclear cell was induced by SEB in vitro. Also, human pulmonary artery endothelial cell (HPAEC) was cocultured with SEB-activated T cell supernatant and secretion of chemokine was examined.To further determine whether these changes also extended to increased activity on chemoattraction and adherence,the transwell inserts was used in chemoattraction assays. Moreover, endothelial cell damage and protection effect of Met-Rantes were deteced in SEB-activated T cell and HPAEC coculture model.We anticipated that thess finding will aid in elucidating the mechanisms of Staphylococcus aureus sepsisMETHODS:Part 1.The change of T cell activated by SEB in vitroPBMC was isolated from heathy adult donor and cutured in vitro with 1,10,100ng/ml SEB stimulation. The transcription and expression of CCR2,CCR3,CCR5 were meseured with Real-time fluorescent quantitation polymerase chain reaction (RT-PCR) and fluorescence-activated cell sorter (FACs) analysis of T cell. Proliferation of T cell was detected by MTS assay at 1d,3d,5d,7d,9d after SEB stimulation and the Vβrepertoire was determined by flowcytometry after stained with T cell Vβfamily antibodies. While cytokines in supernatant were meseured with ELISA at 6,12,24,48,72h after SEB stimulation.All changes were integrated according to time of SEB stimulation to determin which alternation is the most possible marker of superantigen effects.Part 2.The impact of SEB-activated T cell on HPAEC and the possible mechanisms1,HPAEC was cocultured with SEB-activated T cell supernatant and secretion of MCP-1,MIP-1α,Rantes from HPAEC was examined with ELISA. The transwell inserts was used in chemoattraction assays to further determine whether the chemokines from HPAEC plus increased expression of chemokine receptor on T cell extended to increased activity on chemoattraction and adherence. To evaluate the role of chemokine receptor on T cell in the course of chemoattraction and adherence, the HPAEC-adherent T cell monolayer was dispersed with trypsin and prepared for flowcytometry with CCR5/CD4 or CCR5/CD8 monoclonal antibodies incubation; and the antagonistic effect of Met-Rantes was investigated.2,HPAEC was cocultured with 10ng/ml SEB,T cell supernatant induced with 10ng/ml SEB for 3 days and T cell activated with 10ng/ml SEB for 3d respectively.HPAEC damage was monitored by microscopy and TUNEL assay over the course of 48 hours, while HPAEC activation was investigated by comparing cell-surface expression levels of adhesion molecules (ICAM-1,P-seletion) with cell immunohistochemistry assay and AQP-1 transcription with RT-PCR among cell groups. RESULTS:Part 1. The change of T cell activated by SEB in vitro1,A large amount of T blast cell aggregation was detected by inverted microscopy after PBMC was induced with SEB , which was distinguished from control group. But this classic superantigen phenomenon coud hardly be quantitatively measured in the early phase of T cell proliferation. Athough T cell proliferation became more detectable according to SEB density, statistics difference in T cell quantity between SEB-treated groups and control group could be disclosed as late as at 5th day through MTS assay( 1ng/ml SEB group 0.4812±0.0253 vs 0.3172±0.0121,P<0.01).Samely, Vβrepertoire skewing on TCR Vβ3,TCR Vβ17,TCRVβ14 can be discovered by FACs at least at 5th day .2,RT-PCR and FACs test of CCR2,CCR3,CCR5 on T cell showed a significant decrease in expression of CCR2 and a sharp increase in expression of CCR5 among all SEB groups. Hower, there is no significant differences in expression of CCR3 between control group and SEB groups.3,ELISA assay of T cell cytokine showed density of 4 kinds of tested cytokines in supernatant kept relatively constant in control group while changed markedly in SEB groups: (1) The secretion of IL-2 present a dose-dependant model after 12h, peaked nealy at 24h and kept relatively constant to 72h in all SEB groups; at 72th h, the level of IL-2 in 1ng/ml SEB group was higher than control group. (25.4±2.08 vs 19.6±2.11,t=2.875; P<0.05).(2) TNF-αshowed no change in early 6h, the first increase of TNF-αsecretion happened in 100ng/ml SEB group at 12h.(457.5±28.0 vs 350.2±21.4,t=4.848; P<0.01).then increased sharply so much that the level of TNF-αincreased 7 fold ; However, the notably increase could only be tested at 48h in 1ng/ml SEB group and 10ng/ml SEB group. (3) The secretion of IFN-γis dose-dependant also, moreover, all SEB groups showed a markely increase as early as at 12h and rised quikly hence. (4).As a marker of"Th2"cytokine, IL-10 level also rised according to density of SEB at 24h that even in 1ng/ml SEB group showed a statistic increase. (160.7±10.8 vs 97.6±11.3, t=6.966 P<0.01).Part 2.The impact of SEB-activated T cell on HPAEC and the possible mechanisms1,Three kinds of tesed chemokins showed a time-dependant increase in all supernatant groups, and Rantes present two-phase secretion model: in early 6h it increase swiftly and relatively slowely from 12-72h.Copared with 10ng/ml SEB treated alonly , HPAEC secreat more MCP-1,MIP-1α,Rantes in SEB-activated T cell supernatant groups ,especially MCP-1 and Rantes: MCP-1 level rised 17 fold and 22 fold (1200±68 vs 69±6;6500±500 vs 303±8)at 6h and 72h respectively in 1×T supernatant group, while Rantes level rised 33 fold and 140 fold (3600±138 vs 114±7;15700±1300 vs116±12)at 6h and 72h respectively. Different from MIP-1α, MCP-1 and Rantes still showed a density-dependant increase in all supernatant groups, but even SEB-activated T cell supernatant was diluted to 1/100, MCP-1 and Rantes level was higher than SEB group. MIP-1αsecretion showed more time-dependant than dose-dependant that it changed lately at 48h in 1/100 T cell supernatant group.( 230±11 vs 75±10,P<0.01).2,In Transwell insert chemoattraction assays, when non-SEB-activated T cell was loaded in upper well chambers and T cell cytokine induced HPAEC supernant was loaded in lower well chambers, T cell adherent to polycarbonate membrane increased markely (71.00±6.13 vs 16.50±2.50;P<0.01); when SEB-activated T cell was loaded in upper well chambers and control HPAEC supernant was loaded in lower well chambers, T cell adherent to polycarbonate membrane also increased(50.88±6.56 vs 16.50±2.50;P<0.01); when SEB-activated T cell was loaded in upper well chambers and T cell cytokine induced HPAEC supernant was loaded in lower well chambers, T cell adherent to polycarbonate membrane increased at most, (86.38±14.50 vs 16.50±2.50;P<0.01),but if Met-Rantes was loaded in lower well chambers, T cell adherent to polycarbonate membrane decreased markely.(42.38±6.67 vs 86.38±14.50;P<0.01).Even thus, the adherent T cell amount is more than control group. (42.38±6.67 vs 6.50±2.50;P<0.01).3,When 10ng/ml SEB activated T cell was cocultured with HPAEC, more originally suspended cultured T cell adhered to HPAEC monolayer (15.5±1.08 vs 1.60±0.22;P<0.01)whrease the cell adhesion ration decreased markely in 1ug/ml Rantes group.(4.39±0.66 vs1.60±0.22;P<0.01).FACs test of HPAEC-adherent T cell showed CCR5+/CD4+ and CCR5+/CD8+ account to 52.30±4.45% and 48.05±3.66%, which increased over 3 fold (52.30±4.45% vs 14.8±1.02%)and 4 fold( 48.05±3.66 %vs 12.6±1.31%)compared with 100ng/ml SEB activated T cell.4,HPAEC experienced great change in cell size and appearance when 10ng/ml SEB activated T cell was cocultured with HPAEC,the nucleus swelled and cell appearance became irregular; cell gaps expanded and cell obscission can be deteced from 24h. TUNEL test showed a sharp increase of cell appotosis ratio in this group. (32.50±4.50 vs3.50±0.50,P<0.01). what is more, the smaller nucleur of adherent T cell could be discovered near apoptosis HPAEC in many fields of view by microsopy. However, cell appotosis ratio remained unchanged in SEB group,T cell group and control group.Athough a increased tendency was observed, cell appotosis ratio was no significant different in supernatant groups from controls.(5.25±1.25vs3.50±0.50,P=0.089).Copared with superantigen groups, cell appotosis ratio decreased in Met-Rantes groups. (10.33±0.66vs32.50±4.50,P<0.01)5,RT-PCR assay of mRNA of AQP1 showed that different from other groups, the expression of AQP1 of HPAEC only increased in superantigen groups which up-regulated to 4.87 fold. Compared with superantigen groups, the expression of AQP1 decreasded in Met-Rantes groups(.2.76±0.38 vs 4.87±0.60,P<0.01)6,Cell immunohistochemistry assay showed an up-regulation of ICAM-1 expression on HPAEC both in superantigen groups and supernantant groups, especially in superantgen groups deeply dying cycles of ICAM-1 were discovered in view fields through high power lens. P-selectin also up-regulated in the both groups compared with controls, but there is no significant difference between the both groups.Copared with superantigen groups, both expression of ICAM-1 and P-selectin were inhibit by Met-Rantes.CONCLUSIONS :1,SEB was able to induce T cell proliferate in early phase in vitro, but MTS assay and FACs with TCRβfamily antibodies failed to detected this classic"superantigen"phenomenon in early phase because only 2 among 25 subgroups of T cell ,that was TCR Vβ3,TCR Vβ17 and TCR Vβ14 subgoups were proliferating.2,Different from previous reports of"Th1 storm", we finded SEB-activated T cell released not only Th1 type cytokine but also Th2 type cytokine. Besides classic Th1 type cytokine ,IFN-γ; the marker of Th2 type cytokine, IL-10 ; T cell proliferation cytokine IL-2 and proinflammatory cytokine TNF-αalso sheded more depending on SEB density and inducing time.3,Characteristic change happended earlier in cheomine receptor on T cell after T cell was induced by SEB, CCR2 down-regulated whereas CCR5 up-regulated, which suggested CCR5 coud be regarded as a marker of T cell activation with superantigen. 4,Both up-regulation of cheomokine receptor on T cell and increase of MCP-1,MIP-1αand Rantes secreation from HPAEC attributed to increasing chemoattraction and adherence of SEB activated T cell to HPAEC ,especially CCR5 and Rantes.5,Icreasing chemoattraction and adherence of SEB activated T cell to HPAEC could damage and activate HPAEC simultaneously, this effect was owing to direct T cell adherence more than cytokine release from activated T cell on HPAEC.Thus, we should pay more attention on this direct T cell effect in mechanisms of Staphylococcus aureus sepsis.6,Met-Rantes could inhibit chemoattraction and adherence of SEB activated T cell to HPAEC and consequently inhibit the damage of HPAEC, which provide a new therapeutic strategy in Staphylococcus aureus sepsis.