Simon S I, Chambers J D, Butcher E, Sklar L A
Department of Pathology, University of New Mexico School of Medicine, Albuquerque 87131.
J Immunol. 1992 Oct 15;149(8):2765-71.
Neutrophil aggregation in response to formyl peptide was analyzed in blood and isolated cells by fluorescence flow cytometry. The isolated leukocyte aggregates and the leukocytes in blood were identified with the vital nucleic acid stain LDS-751. This method enabled us to discriminate nucleated cells from other blood cells and to detect granulocyte aggregates without isolation or E lysis. Cells isolated in the absence of endotoxin retained the characteristics of cells in blood and exhibited similar aggregation kinetics and dose-response to formyl peptide. We show that it is possible to analyze epitope expression in blood with homogeneous flow cytometric assays and that carefully isolated neutrophils retain the expression characteristics of those in blood. The expression of CD18 was at its lowest levels in unstimulated cells, while the rate of formyl peptide stimulated aggregation was most rapid in these cells. Aggregation in isolated cells as well as blood preceded an increase in receptor expression. After stimulation, L-selectin expression decreased in both blood and isolated cells over a time frame similar to disaggregation. The aggregation response in blood was blocked by pretreatment with antibody to CD18 over a concentration range consistent with the amount of antibody bound. Aggregation was also blocked in isolated cells and blood by antibodies DREG-200 and DREG-56 to L-selectin, but not by isotype controls or anti-LFA-1. The results are discussed in terms of the roles of adhesive receptor expression and recognition in neutrophil aggregation. The methods validated here permit linkage between isolated cells and in vivo studies.
通过荧光流式细胞术分析血液和分离细胞中对甲酰肽的中性粒细胞聚集情况。使用活性核酸染料LDS - 751鉴定血液中的分离白细胞聚集体和白细胞。该方法使我们能够区分有核细胞与其他血细胞，并在不进行分离或红细胞裂解的情况下检测粒细胞聚集体。在无内毒素条件下分离的细胞保留了血液中细胞的特征，并表现出相似的聚集动力学和对甲酰肽的剂量反应。我们表明，利用均相流式细胞术分析血液中的表位表达是可行的，并且仔细分离的中性粒细胞保留了血液中中性粒细胞的表达特征。CD18的表达在未刺激细胞中处于最低水平，而这些细胞中甲酰肽刺激的聚集速率最快。分离细胞和血液中的聚集先于受体表达增加。刺激后，血液和分离细胞中的L - 选择素表达在与解聚相似的时间范围内下降。用与结合抗体量一致的浓度范围内的抗CD18抗体预处理可阻断血液中的聚集反应。抗L - 选择素的抗体DREG - 200和DREG - 56也可阻断分离细胞和血液中的聚集，但同型对照或抗LFA - 1抗体则不能。从粘附受体表达和识别在中性粒细胞聚集中的作用方面对结果进行了讨论。这里验证的方法允许将分离细胞的研究与体内研究联系起来。
