Crucian Brian, Nelman-Gonzalez Mayra, Sams Clarence
Wyle Laboratories, Space Phystiology and Countermeasures Department, NASA-Johnson Space Center, Human Adaptation and Countermeasures Office (SK3), Houston, TX 77058, USA.
Clin Vaccine Immunol. 2006 Mar;13(3):403-8. doi: 10.1128/CVI.13.3.403-408.2006.
Adhesion molecules are important for leukocyte endothelial attachment and migration to sites of inflammation. The LFA-1 (CD11a and CD18) integrin molecule is constitutively expressed on the T-cell surface. Following T-cell activation, a rapid conformational change of LFA-1 to an "adhesive" state occurs, allowing LFA-1 binding to intracellular cell adhesion molecule type 1 (ICAM-1)-expressing targets, such as antigen-presenting cells. For this study, a rapid flow cytometry method for the quantitation of LFA-1-adhesive T cells following activation was developed. Purified ICAM-1 was bound to 4.5-microm-diameter beads. Following peripheral blood mononuclear cell activation culture (phorbol myristate acetate and ionomycin), the cells were incubated with the ICAM-1 beads, which allowed attachment to occur. The T cell-bead complexes were then resolved from unbound T cells by flow cytometry. Multicolor analysis allowed a complete phenotypic analysis of the adhesive T-cell subsets. Experimental controls indicated that the T cell-bead attachment was LFA-1 and ICAM-1 specific. Very little binding between unactivated T cells and ICAM beads or between activated T cells and plain beads was observed. The kinetics of the response was extremely rapid, with nearly maximal numbers of adhesive T cells observed following 5 min of activation. Scanning electron microscopy analysis was used to characterize legitimate bead-cell binding. By using multicolor cytometry, the responding adhesive T-cell population was usually identified as a distinct subset of T cells with the following phenotype: CD3+ CD4+ or CD8+ CD19- CD16- CD45RO+ CD62L+ CD27+ CD57-. A rapid and simple method for the scoring of LFA-1-adhesive T cells was developed and may have significant utility for immune function studies.
黏附分子对于白细胞与内皮细胞的附着以及向炎症部位的迁移至关重要。淋巴细胞功能相关抗原-1（LFA-1，由CD11a和CD18组成）整合素分子在T细胞表面组成性表达。T细胞激活后，LFA-1会迅速发生构象变化，转变为“黏附性”状态，使LFA-1能够与表达细胞间黏附分子-1（ICAM-1）的靶细胞结合，如抗原呈递细胞。在本研究中，开发了一种用于定量激活后LFA-1黏附性T细胞的快速流式细胞术方法。将纯化的ICAM-1与直径4.5微米的珠子结合。外周血单个核细胞激活培养（使用佛波酯肉豆蔻酸酯和离子霉素）后，将细胞与ICAM-1珠子一起孵育，使其发生附着。然后通过流式细胞术将T细胞-珠子复合物与未结合的T细胞分离。多色分析能够对黏附性T细胞亚群进行完整的表型分析。实验对照表明，T细胞与珠子的附着具有LFA-1和ICAM-1特异性。未激活的T细胞与ICAM珠子之间或激活的T细胞与普通珠子之间几乎没有观察到结合。反应动力学极其迅速，激活5分钟后即可观察到几乎最大数量的黏附性T细胞。使用扫描电子显微镜分析来表征合法的珠子-细胞结合。通过使用多色细胞术，应答的黏附性T细胞群体通常被鉴定为具有以下表型的独特T细胞亚群：CD3 + CD4 +或CD8 + CD19 - CD16 - CD45RO + CD62L + CD27 + CD57 -。开发了一种快速简单的LFA-1黏附性T细胞评分方法，可能对免疫功能研究具有重要用途。
