發炎反應發生時，血管中的白血球會由快速流動、滾動、黏著於內皮細胞，到最後移行(transmigration)至特定部位。這樣的過程在人體免疫功能上，扮演著很重要的角色，且發炎反應即是人體用來抵禦致病原入侵的第一道防線。白血球與內皮細胞間的交互作用，必須依靠多種細胞黏著分子的幫忙，其中包括selectin、integrin與intercellular adhesion molecules。ICAM-1屬於immunoglobulin superfamily，具有五個extracellular Ig-like domains。在發炎反應中，許多刺激(如TNF-α、INF-γ、IL-1等)都可以增強血管內皮細胞ICAM-1分子的表現。ICAM -1最主要可與白血球上LFA-1結合，完成細胞黏著步驟。然而，發炎內皮細胞與白血球黏著、移行的調控機制，仍有許多未知。在此我們利用實驗室之前發現的抗ICAM-1抗體，EN2與I9來幫助我們探討這些問題。首先我們發現，EN2或I9均可促使白且球與內皮細胞之間的黏著。接著我們更進一步的去觀察，發現EN2與I9也會抑制白血球的移行。也就是說，當EN2與I9增強白血球與內皮細胞的黏著，由於緊密黏著的增加，也使得移行的白血球數目減少。由於ICAM-1可利用不同的domain與其配位體結合，於是我們製備六種不同truncated form的ICAM-1，以便幫助我們觀察抗ICAM-1抗體結合的辨識部位。另外，我們也想利用flow chamber建立流動系統，希望可以在一個穩定的狀態下觀察白血球黏著情形，減少adhesion assay清洗步驟的人為操作誤差。

　　發炎反應中，另一種相當重要的黏著分子是integrin。根據過去的研究顯示，integrin的活性與其本身蛋白質構造有密切關係。Integrin會利用可逆性的氧化還原反應，來改變結構狀態，以達到活性開關的轉換。目前有許多關於血小板integrin的研究證實，ROS可直接在integrin的cystine-rich domain上進行氧化還原，如此結構的改變會造就integrin活性的變化。而α4 integrin也含有相似的domain，所以我們想知道這類的integrin是否也可藉由ROS的調控，來達到活性變換的效果。依據實驗室之前的研究發現，H2O2可調控VLA-4與VCAM-1的結合。高濃度的H2O2可抑制VLA-4與VCAM-1的結合，但低濃度的H2O2則有增進結合的效果。我們知道，VLA-4與VCAM-1在白血球的滾動上扮演重要角色。所以接下來要用flow chamber從白血球滾動的狀態，來觀察H2O2對VLA-4與VCAM-1結合的影響。最後我們發現，H2O2在高濃度的狀態下，滾動的白血球數目會減少;然而在低濃度的情況下，滾動白血球的數目會增加。確實利用流動測試系統觀察到H2O2濃度的不同，對VLA-4與VCAM-1結合會有不同的影響調控。

　Leukocytes adhere to endothelium then transmigrate through blood vessels and traffic to different tissues. This process is important for host defense mechanisms and inflammatory response. The adhesion between leukocyte and endothelium is mediated by several cell surface receptors, including selectins, integrins, and immunoglobulin (Ig) superfamily cell-adhesion molecules (CAMs). Intercellular adhesion molecule-1 (ICAM-1) is an immunoglobulin (Ig) superfamily molecule which contains five Ig-like domains. Many inflammatory mediators activate endothelial cell expression of ICAM-1, which serves as a ligand for the integrin, LFA-1. The regulation mechanisms of leukocyte adhesion and transmigration in the inflamed endothelium mediated by integrein-Ig superfamily molecule thus are critical for immune responses. We previously prepared two monoclonal antibodies, EN2 and I9, which bind to ICAM-1. We found that human umbilical vein endothelial cells (HUVECs) treated with EN2 or I9 have enhanced adhesion between leukocytes and HUVECs. We also found that EN2 and I9 decrease leukocyte transmigration through HUVEC monolayers. We thus hypothesized that the adhesion ability changed by EN2 or I9 is involved in the regulation of cell transmigraton in inflammatory tissues. Based on the hypothesis that EN2 and I9 may bind to different epitopes on ICAM-1, we prepared six truncated forms of ICAM-1 (ICAM-1 five domains and ICAM-1 lacking domain 1, domain 1-2, domain 1-3, domain 4-5, domain 5) to map the binding sites of EN2 and I9 on ICAM-1. A flow-chamber assay system was established to test the adhesion function of leukocytes which may be modulated by these recombinant ICAM-1 molecules.
　We also investigated the modulation of integrins in inflammation. Recent studies showed that conformational changes cause activation or deactivation of integrins. Reactive oxygen species (ROS) can modulate the integrin activity by reversible redox reaction. We thus tested whether ROS change the adhesion states of leukocytes in flow chamber systems. In line with our previous studies, we found that H2O2 can modulate the interaction between VLA-4 and VCAM-1. We selected wall shear stress 1 dyne/cm2 to mimic normal physiological microvascular environments. In a lower concentration (10M), H2O2 enhanced VLA-4 binding to VCAM-1, while in a higher concentration (250M), H2O2 decreased VLA-4 binding to VCAM-1. These results support the hypothesis that ROS modulate the integrin-Ig superfamily molecule interactions and is critical for leukocyte trafficking in inflammatory tissues.

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