Symon F A, Walsh G M, Watson S R, Wardlaw A J
Department of Respiratory Medicine, University of Leicester Medical School, Glenfield General Hospital, United Kingdom.
J Exp Med. 1994 Jul 1;180(1):371-6. doi: 10.1084/jem.180.1.371.
Tissue eosinophilia is a characteristic feature of a number of inflammatory diseases including asthma and nasal polyposis. Eosinophil migration into tissues is controlled in part by interactions between eosinophil adhesion receptors and counter-structures on the vascular endothelium. To determine the receptors used by eosinophils to adhere to vascular endothelium in allergic inflammation we have adapted the Stamper-Woodruff frozen section assay (FSA) to study eosinophil adhesion to nasal polyp endothelium. Immunohistology indicated that intercellular adhesion molecule 1 (ICAM-1), E-selectin and P-selectin were well expressed by nasal polyp endothelium, whereas expression of vascular cell adhesion molecule 1 (VCAM-1) was weak or absent. Unstimulated human peripheral blood eosinophils adhered specifically to nasal polyp endothelium. Adherence was temperature and divalent cation-dependent and saturable at cell densities > 5 x 10(6) cells/ml. Eosinophil adhesion was almost completely inhibited by a monoclonal antibody (mAb) against P-selectin and by a chimeric molecule consisting of the Fc portion of human IgG and the lectin binding domain of P-selectin, which binds to the P-selectin ligand on leucocytes. Anti-Mac-1 mAb partially inhibited eosinophil adhesion whereas mAb against E-selectin, L-selectin, ICAM-1, VCAM-1, very late activation antigen 4, and lymphocyte function-associated antigen 1 had no effect. P-selectin is stored in intracellular granules within the endothelial cell and in vitro is only transiently expressed. To determine if P-selectin was expressed on the membrane of the nasal polyp endothelium we compared P-selectin expression in normal skin and nasal polyps after acetone fixation, which permeabilizes cells, and paraformaldehyde, which only allows staining of membrane expressed receptors. In the skin, good expression was seen with acetone fixation but no expression was seen after paraformaldehyde treatment, whereas in nasal polyps, similar expression was observed with both fixatives. In addition immunofluorescence with confocal microscopy demonstrated lumenal staining of nasal polyp endothelium indicating that P-selectin was located on the surface of endothelial cells while in skin only an intracellular granular distribution was apparent. Lastly, whereas eosinophils bound consistently to nasal polyp endothelium, no binding was observed to blood vessels in normal skin further supporting the idea that eosinophils were binding to membrane expressed and not intracellular P-selectin. The importance of P-selectin in eosinophil adhesion to nasal polyp endothelium suggests that P-selectin antagonists may be effective at inhibiting eosinophil accumulation at sites of allergic inflammation.
组织嗜酸性粒细胞增多是包括哮喘和鼻息肉病在内的多种炎症性疾病的特征性表现。嗜酸性粒细胞向组织的迁移部分受嗜酸性粒细胞黏附受体与血管内皮细胞上相应结构之间相互作用的控制。为了确定嗜酸性粒细胞在过敏性炎症中黏附于血管内皮细胞所使用的受体，我们采用了斯坦珀 - 伍德拉夫冰冻切片试验（FSA）来研究嗜酸性粒细胞与鼻息肉内皮细胞的黏附。免疫组织学表明，鼻息肉内皮细胞高表达细胞间黏附分子1（ICAM - 1）、E - 选择素和P - 选择素，而血管细胞黏附分子1（VCAM - 1）表达较弱或无表达。未受刺激的人外周血嗜酸性粒细胞特异性黏附于鼻息肉内皮细胞。黏附呈温度和二价阳离子依赖性，在细胞密度>5×10⁶细胞/ml时达到饱和。抗P - 选择素单克隆抗体（mAb）和一种由人IgG的Fc部分与P - 选择素的凝集素结合域组成的嵌合分子几乎完全抑制了嗜酸性粒细胞的黏附，该嵌合分子可与白细胞上的P - 选择素配体结合。抗Mac - 1 mAb部分抑制嗜酸性粒细胞黏附，而抗E - 选择素、L - 选择素、ICAM - 1、VCAM - 1、极晚期活化抗原4和淋巴细胞功能相关抗原1的mAb则无作用。P - 选择素储存于内皮细胞的胞内颗粒中，在体外仅短暂表达。为了确定P - 选择素是否在鼻息肉内皮细胞膜上表达，我们比较了丙酮固定（可使细胞通透）和多聚甲醛固定（仅能对膜表达的受体进行染色）后正常皮肤和鼻息肉中P - 选择素的表达情况。在皮肤中，丙酮固定后可见良好表达，而多聚甲醛处理后未见表达；而在鼻息肉中，两种固定剂处理后观察到相似的表达。此外，共聚焦显微镜免疫荧光显示鼻息肉内皮细胞腔面染色，表明P - 选择素位于内皮细胞表面，而在皮肤中仅可见胞内颗粒分布。最后，虽然嗜酸性粒细胞始终与鼻息肉内皮细胞结合，但未观察到与正常皮肤血管的结合，这进一步支持了嗜酸性粒细胞是与膜表达而非胞内P - 选择素结合的观点。P - 选择素在嗜酸性粒细胞与鼻息肉内皮细胞黏附中的重要性表明，P - 选择素拮抗剂可能有效抑制嗜酸性粒细胞在过敏性炎症部位的积聚。
