Bellinati-Pires R, Salgado M M, Hypolito I P, Grumach A S, Carneiro-Sampaio M M
Seção de Imunologia, Instituto Adolfo Lutz, São Paulo, Brasil.
J Investig Allergol Clin Immunol. 1995 Nov-Dec;5(6):337-42.
The evaluation of phagocytic and microbicidal activities of the blood neutrophils has been recognized as one of the important tools for investigating phagocytic dysfunctions in patients with recurrent infections. In the present study, these activities were examined in neutrophils and monocytes from healthy adults and patients affected by primary phagocytic dysfunctions by using a modified fluorochromic microbicidal assay, discriminating simultaneously the extracellular adherence, ingestion and intracellular killing of Staphylococcus aureus Cowan I. The assay employs acridine orange staining, as described in Bellinati-Pires et al. (1989) (AO assay), but was modified by the addition of an alternative leukocyte treatment with 0.5 U/ml of lysostaphin (LS) for 5 min at 37 degrees C, after phagocytosis (AO-LS assay). The LS treatment was standardized to eliminate staphylococci adhered to the outer surface of the phagocytes without affecting the determination of intracellular live or dead bacteria, as demonstrated in normal neutrophils and monocytes. Our purpose in this study was to compare AO and AO-LS assays in order to evaluate the effect of LS on the determination of actually ingested staphylococci and to provide a means for improving the fluorochromic assay for detecting phagocytic defects, as well as bactericidal disturbances. By using the AO-LS assay, decreased ingestion of staphylococci by neutrophils in Chediak-Higashi Syndrome (CHS) was demonstrated. However, increased staphylococci adherence, as well as ingestion, was observed in neutrophils or monocytes from chronic granulomatous disease (CGD) patients, comparing AO and AO-LS assays. Bactericidal defect, which is a common feature in CHS and CGD, was detected in neutrophils or monocytes in both assays. We emphasize that such alterations were deduced by comparing the patients' results with those obtained from their respective normal controls and with the normal range of values previously established for 160 healthy adults. No alteration was observed in hyper IgE syndrome phagocytes. Despite the possible penetration of LS into the leukocytes, as stated in other studies, we concluded that a short period of phagocyte incubation with this enzyme increased the sensitivity of the fluorochromic assay to detect phagocytic defect without affecting the determination of the bactericidal activity. Moreover, comparations between AO and AO-LS assays may be important in the study of the initial pathways of staphylococci phagocyte interaction, including adherence by non-phagocytic receptors.
血液中性粒细胞吞噬和杀菌活性的评估已被视为研究反复感染患者吞噬功能障碍的重要工具之一。在本研究中，通过使用改良的荧光杀菌试验，对健康成年人以及受原发性吞噬功能障碍影响的患者的中性粒细胞和单核细胞中的这些活性进行了检测，该试验同时区分了金黄色葡萄球菌考恩I型的细胞外黏附、摄取和细胞内杀伤情况。该试验采用了贝利纳蒂 - 皮雷斯等人（1989年）所述的吖啶橙染色法（AO试验），但在吞噬作用后（AO - LS试验）进行了改良，即在37摄氏度下用0.5 U/ml溶葡萄球菌素（LS）对白细胞进行5分钟的替代处理。如在正常中性粒细胞和单核细胞中所证实的那样，LS处理经过标准化，以消除黏附在吞噬细胞外表面的葡萄球菌，而不影响细胞内活菌或死菌的测定。本研究的目的是比较AO试验和AO - LS试验，以评估LS对实际摄取的葡萄球菌测定的影响，并提供一种改进荧光试验以检测吞噬缺陷以及杀菌干扰的方法。通过使用AO - LS试验，证实了切迪阿克 - 希加综合征（CHS）患者中性粒细胞对葡萄球菌的摄取减少。然而，比较AO试验和AO - LS试验时，在慢性肉芽肿病（CGD）患者的中性粒细胞或单核细胞中观察到葡萄球菌黏附以及摄取增加。在两种试验中，均在中性粒细胞或单核细胞中检测到了CHS和CGD中常见的杀菌缺陷。我们强调，这些改变是通过将患者的结果与各自正常对照的结果以及先前为160名健康成年人确定的正常数值范围进行比较而推断出来的。在高IgE综合征吞噬细胞中未观察到改变。尽管如其他研究所述，LS可能会渗透到白细胞中，但我们得出结论，用这种酶对吞噬细胞进行短时间孵育可提高荧光试验检测吞噬缺陷的敏感性，而不影响杀菌活性的测定。此外，AO试验和AO - LS试验之间的比较在研究葡萄球菌与吞噬细胞相互作用的初始途径（包括通过非吞噬性受体的黏附）中可能很重要。
