Hakkert B C, Rentenaar J M, Van Aken W G, Roos D, Van Mourik J A
Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam.
Eur J Immunol. 1990 Dec;20(12):2775-81. doi: 10.1002/eji.1830201236.
Leukocyte adhesion to endothelial cells and migration into the subendothelial matrix was studied with a three-dimensional model system, consisting of human endothelial cells cultured on a loose collagen matrix. We developed a new method to separate the endothelial cell monolayer and adhering leukocytes, from the subendothelial matrix, allowing simultaneous analysis of leukocyte adhesion and transendothelial migration. Monocytes adhered more avidly to untreated endothelial cells than did neutrophils (2.5 +/- 0.3 vs. 1.0 +/- 0.2 leukocytes per endothelial cell). Only a small fraction (10%-20%) of these leukocytes migrated into the subendothelium. Pretreatment of endothelial cells with interleukin 1 (IL 1) enhanced adhesion (20%), but not migration of monocytes. In contrast, neutrophil adhesion was markedly and in a time-dependent manner increased by IL 1 treatment (i.e. 200% after 6 h and 110% after 24 h of IL 1 treatment). Moreover, IL 1 pretreatment enhanced neutrophil migration twofold. Activation of leukocytes with formyl-methionyl-leucyl-phenylalanine (fMLP) enhanced both monocyte and neutrophil adhesion, but did not affect leukocyte migration. Under all conditions, monocyte adhesion was only partly (30%-40%) inhibited by monoclonal antibodies (mAb) against the common beta subunit of the leukocyte-cell adhesion molecules (LeuCAM: CD18) and 25%-30% by mAb against the alpha subunit of LFA-1 (CD11a). In contrast, mAb against the alpha subunits of Mac-1 (CD11b) and p150.95 (CD11c) were hardly effective. fMLP-mediated neutrophil adhesion was reduced to below baseline levels by anti-LeuCAM (CD18) mAb, whereas the LeuCAM contribution in IL 1-mediated neutrophil adhesion was less pronounced and varied in time. IL 1-mediated neutrophil migration, however, was completely blocked by anti-LeuCAM mAb. fMLP-mediated neutrophil adhesion was inhibited by mAb against the alpha subunits of Mac, while mAb against the alpha subunits of LFA-1 and Mac-1 both reduced IL 1-mediated adherence. In summary, we describe a novel leukocyte adhesion/migration method and demonstrate that the contribution of the LeuCAM complex in leukocyte-endothelium interaction varies depending on cell type and stimulus used.
利用一种三维模型系统研究白细胞与内皮细胞的黏附以及向内皮下基质的迁移，该系统由培养在疏松胶原基质上的人内皮细胞组成。我们开发了一种新方法，可将内皮细胞单层和黏附的白细胞与内皮下基质分离，从而能够同时分析白细胞黏附和跨内皮迁移。单核细胞比中性粒细胞更 avidly 地黏附于未处理的内皮细胞（每个内皮细胞分别为 2.5±0.3 和 1.0±0.2 个白细胞）。这些白细胞中只有一小部分（10%-20%）迁移到内皮下。用白细胞介素 1（IL 1）预处理内皮细胞可增强黏附（20%），但不增强单核细胞的迁移。相比之下，IL 1 处理可显著且呈时间依赖性地增加中性粒细胞的黏附（即 IL 1 处理 6 小时后增加 200%，24 小时后增加 110%）。此外，IL 1 预处理使中性粒细胞迁移增加两倍。用甲酰甲硫氨酰亮氨酰苯丙氨酸（fMLP）激活白细胞可增强单核细胞和中性粒细胞的黏附，但不影响白细胞迁移。在所有条件下，针对白细胞-细胞黏附分子共同β亚基（LeuCAM：CD18）的单克隆抗体（mAb）仅部分（30%-40%）抑制单核细胞黏附，针对淋巴细胞功能相关抗原 1（LFA-1）α亚基（CD11a）的 mAb 抑制 25%-30%。相比之下，针对巨噬细胞-1（Mac-1，CD11b）和 p150.95（CD11c）α亚基的 mAb 几乎无效。抗 LeuCAM（CD18）mAb 可将 fMLP 介导的中性粒细胞黏附降低至基线水平以下，而 LeuCAM 在 IL 1 介导的中性粒细胞黏附中的作用不太明显且随时间变化。然而，抗 LeuCAM mAb 可完全阻断 IL 1 介导的中性粒细胞迁移。抗 Macα亚基的 mAb 可抑制 fMLP 介导的中性粒细胞黏附，而抗 LFA-1 和 Mac-1α亚基的 mAb 均可降低 IL 1 介导的黏附。总之，我们描述了一种新型的白细胞黏附/迁移方法，并证明 LeuCAM 复合物在白细胞-内皮细胞相互作用中的作用因细胞类型和所用刺激而异。
