Srinivas U, Dohlsten M, Kalland T, Lundblad A
Department of Clinical Chemistry, Faculty of Health Sciences, Hospital Pharmacy, University Hospital, Linköping, Sweden.
Scand J Immunol. 1998 Aug;48(2):127-35. doi: 10.1046/j.1365-3083.1998.00322.x.
In-vivo exposure to the bacterial superantigen Staphylococcal enterotoxin-A (SEA) induces an inflammatory response characterized by rapid extravasation of leucocytes and release of excessive amounts of cytokines. We have utilized an in-vitro adhesion assay to understand the molecular mechanisms responsible for SEA-induced extravasation of leucocytes. Stimulation of human umbilical cord endothelial cells (HUVEC) with increasing concentrations of recombinant SEA (rSEA) did not influence the in-vitro adhesion of HL-60 cells to HUVEC, whereas stimulation of HUVEC by interleukin (IL)-1beta supported adhesion of HL-60 cells. Increased adhesion of HL-60 cells to HUVEC was noted upon stimulation of endothelium with culture medium obtained from human peripheral blood mononuclear cells (PBM) stimulated with recombinant SEA for 24 (CM-SEA 24 h), 72 (CM-SEA 72 h) and 120 h (CM-SEA 120 h), but not after stimulation with culture medium obtained from control human peripheral blood mononuclear cells (CM), suggesting that soluble factors present in the supernatants play a major role in SEA-induced cell adhesion. While CM-SEA 24 and 72 h induced both a rapid (4 h) and delayed type of adhesion, CM-SEA 120 h only induced a delayed type of adhesion. Stimulation of PBM by SEA resulted in increased levels of IL-1beta, IL-2 and interferon (IFN)-gamma after 24h. Further stimulation for 72-120h resulted in a significant increase in the levels of IL-1beta, IFN-gamma and tumour necrosis factor (TNF). Stimulation of PBM with SEA also resulted in increased levels of soluble and L-selectin in the cell supernatants. Increased cell-surface expression of E-selectin, ICAM-1, HLA-DR and VCAM-1 was detected on HUVEC stimulated with CM-SEA media. While E-selectin and VCAM were induced on HUVEC within a few hours, induction of ICAM and HLA-DR required a longer induction period. Adhesion of HL-60 cells to HUVEC treated with CM-SEA was inhibited by monoclonal antibodies (MoAbs) against both the selectin and integrin families of cell adhesion molecules, suggesting that multiple pathways contribute to SEA-induced leucocyte extravasation. The results suggested that selectin-dependent adhesion was more prominent during the early phase while integrin-induced adhesion occurred at a later stage.
体内暴露于细菌超抗原葡萄球菌肠毒素A（SEA）会引发炎症反应，其特征为白细胞迅速渗出并释放大量细胞因子。我们利用体外黏附试验来了解SEA诱导白细胞渗出的分子机制。用浓度递增的重组SEA（rSEA）刺激人脐静脉内皮细胞（HUVEC），并未影响HL-60细胞与HUVEC的体外黏附，而白细胞介素（IL）-1β刺激HUVEC则促进HL-60细胞的黏附。用重组SEA刺激人外周血单个核细胞（PBM）24小时（CM-SEA 24小时）、72小时（CM-SEA 72小时）和120小时（CM-SEA 120小时）后获得的培养基刺激内皮细胞，HL-60细胞与HUVEC的黏附增加，但用对照人外周血单个核细胞获得的培养基刺激后则未增加（CM），这表明上清液中存在的可溶性因子在SEA诱导的细胞黏附中起主要作用。虽然CM-SEA 24小时和72小时诱导了快速（4小时）和延迟型黏附，但CM-SEA 120小时仅诱导了延迟型黏附。SEA刺激PBM 24小时后，IL-1β、IL-2和干扰素（IFN）-γ水平升高。进一步刺激72 - 120小时导致IL-1β、IFN-γ和肿瘤坏死因子（TNF）水平显著升高。SEA刺激PBM还导致细胞上清液中可溶性和L-选择素水平升高。在用CM-SEA培养基刺激的HUVEC上检测到E-选择素、细胞间黏附分子-1（ICAM-1）、人类白细胞抗原-DR（HLA-DR）和血管细胞黏附分子-1（VCAM-1）的细胞表面表达增加。虽然E-选择素和VCAM在数小时内在HUVEC上被诱导，但ICAM和HLA-DR的诱导需要更长的诱导期。抗细胞黏附分子选择素和整合素家族的单克隆抗体（MoAbs）抑制了HL-60细胞与用CM-SEA处理的HUVEC的黏附，这表明多种途径促成了SEA诱导的白细胞渗出。结果表明，选择素依赖性黏附在早期更为突出，而整合素诱导的黏附发生在后期。
