Hamers M N, Bot A A, Weening R S, Sips H J, Roos D
Blood. 1984 Sep;64(3):635-41.
A mutant strain of Escherichia coli (E. coli ML-35) was used to follow the kinetics of phagocytosis, perforation of the bacterial cell envelope, and inactivation of bacterial proteins by human neutrophils. This particular E. coli mutant strain has no lactose permease, but constitutively forms the cytoplasmic enzyme beta-galactosidase. This implies that the artificial substrate ortho-nitrophenyl-beta-D-galactopyranoside cannot reach the beta-galactosidase unless the bacterial cell envelope has been perforated. Thus, the integrity of the E. coli envelope can be measured simply by the activity of beta-galactosidase with this substrate. Indeed, ingestion of E. coli ML-35 by human neutrophils was followed by perforation of the bacteria (increase in beta-galactosidase activity). Subsequently, the beta-galactosidase activity decreased due to inactivation of the enzyme. With a simple mathematical model and a curve-fitting computer program, we have determined the first-order rate constants for phagocytosis, perforation, and beta-galactosidase inactivation. With 32 normal donors, we found an interdonor variation in these rate constants of 20% to 30% (SD) and an assay variance of 5%. The perforation process closely correlated with the loss of colony-forming capacity of the bacteria. This new assay measures phagocytosis and killing in a fast, simple, and accurate way; it is not hindered by extracellular bacteria. Moreover, this method also measures the postkilling event of inactivation of a bacterial protein, which permits a better detection of neutrophils deficient in this function. The assay can also be used for screening neutrophil functions without the use of a computer program. A simple calculation suffices to detect neutrophil abnormalities. Neutrophils from patients with chronic granulomatous disease (CGD) showed an impaired rate of perforation and thus also of inactivation. Neutrophils from myeloperoxidase-deficient patients or from a patient with the Chediak-Higashi syndrome only showed a retarded inactivation of beta-galactosidase, but normal ingestion and perforation. The role of myeloperoxidase in the killing process is discussed. Although myeloperoxidase does not seem to be a prerequisite for perforation, it probably plays a role in bacterial destruction by normal cells, because the inactivation of bacterial proteins seems strictly myeloperoxidase dependent.
使用大肠杆菌突变株（大肠杆菌ML-35）来追踪人类中性粒细胞的吞噬作用动力学、细菌细胞壁穿孔以及细菌蛋白失活情况。这种特定的大肠杆菌突变株没有乳糖通透酶，但可组成性地形成细胞质酶β-半乳糖苷酶。这意味着除非细菌细胞壁已被穿孔，人工底物邻硝基苯基-β-D-吡喃半乳糖苷无法到达β-半乳糖苷酶。因此，通过该底物的β-半乳糖苷酶活性可简单地测量大肠杆菌细胞壁的完整性。实际上，人类中性粒细胞吞噬大肠杆菌ML-35后，细菌会发生穿孔（β-半乳糖苷酶活性增加）。随后，由于酶失活，β-半乳糖苷酶活性降低。通过一个简单的数学模型和曲线拟合计算机程序，我们确定了吞噬作用、穿孔以及β-半乳糖苷酶失活的一级速率常数。对于32名正常供体，我们发现这些速率常数在供体间的变异为20%至30%（标准差），测定变异为5%。穿孔过程与细菌集落形成能力的丧失密切相关。这种新的测定方法能以快速、简单且准确的方式测量吞噬作用和杀伤作用；它不受细胞外细菌的干扰。此外，该方法还能测量细菌蛋白失活这一杀伤后事件，从而能更好地检测在该功能方面存在缺陷的中性粒细胞。该测定方法也可用于在不使用计算机程序的情况下筛选中性粒细胞功能。一个简单的计算就足以检测中性粒细胞异常。慢性肉芽肿病（CGD）患者的中性粒细胞显示穿孔速率受损，进而失活速率也受损。髓过氧化物酶缺乏患者或患有切-东综合征患者的中性粒细胞仅显示β-半乳糖苷酶失活延迟，但吞噬和穿孔正常。讨论了髓过氧化物酶在杀伤过程中的作用。尽管髓过氧化物酶似乎不是穿孔的先决条件，但它可能在正常细胞对细菌的破坏中起作用，因为细菌蛋白的失活似乎严格依赖于髓过氧化物酶。
