Miyasaki K T, Bodeau A L, Flemmig T F
Section of Oral Biology, UCLA School of Dentistry 90024-1668.
Infect Immun. 1991 Oct;59(10):3760-7. doi: 10.1128/iai.59.10.3760-3767.1991.
The purpose of this study was to determine whether granule fractions of human neutrophils differentially kill Actinobacillus actinomycetemcomitans and Capnocytophaga spp. Granule extracts were subjected to gel filtration, and seven fractions (designated A through G) were obtained. Under aerobic conditions at pH 7.0, representative strains of A. actinomycetemcomitans were killed by fraction D and variably by fraction B. In contrast, the Capnocytophaga spp. were killed by fractions C, D, F, and G. Fractions A (containing lactoferrin and myeloperoxidase) and E (containing lysozyme) exerted little bactericidal activity under these conditions. Anaerobiosis had little effect on the bactericidal activity of fractions D and F but inhibited that of fractions B and C. Electrophoresis, zymography, determination of amino acid composition, and N-terminal sequence analysis revealed that fraction C contained elastase, proteinase 3, and azurocidin. Fraction D contained lysozyme, elastase, and cathepsin G. Subfractions of C and D containing elastase (subfraction C4), a mixture of elastase and azurocidin (subfraction C5), and cathepsin G (subfraction D9) were found to be bactericidal. The bactericidal effects of fraction D and subfraction D9 against A. actinomycetemcomitans was not inhibited by heat inactivation, phenylmethylsulfonyl fluoride, or N-benzyloxycarbonylglycylleucylphenylalanylchloromethyl ketone. We conclude that (i) A. actinomycetemcomitans and Capnocytophaga spp. were sensitive to the bactericidal effects of different neutrophil granule components, (ii) both were sensitive to the bactericidal effects of neutral serine proteases, and (iii) the killing of A. actinomycetemcomitans by cathepsin G-containing fractions was independent of oxygen and neutral serine protease activity.
本研究的目的是确定人类中性粒细胞的颗粒组分对伴放线放线杆菌和二氧化碳嗜纤维菌属的杀伤作用是否存在差异。对颗粒提取物进行凝胶过滤，得到了七个组分（命名为A至G）。在pH 7.0的有氧条件下，伴放线放线杆菌的代表性菌株被组分D杀死，部分被组分B杀死。相比之下，二氧化碳嗜纤维菌属被组分C、D、F和G杀死。组分A（含乳铁蛋白和髓过氧化物酶）和组分E（含溶菌酶）在这些条件下几乎没有杀菌活性。厌氧环境对组分D和F的杀菌活性影响不大，但抑制了组分B和C的杀菌活性。电泳、酶谱分析、氨基酸组成测定和N端序列分析表明，组分C含有弹性蛋白酶、蛋白酶3和天青杀素。组分D含有溶菌酶、弹性蛋白酶和组织蛋白酶G。发现含有弹性蛋白酶的C和D亚组分（亚组分C4）、弹性蛋白酶和天青杀素的混合物（亚组分C5）以及组织蛋白酶G（亚组分D9）具有杀菌作用。组分D和亚组分D9对伴放线放线杆菌的杀菌作用不受热灭活、苯甲基磺酰氟或N-苄氧羰基甘氨酰亮氨酰苯丙氨酰氯甲基酮的抑制。我们得出结论：（i）伴放线放线杆菌和二氧化碳嗜纤维菌属对不同中性粒细胞颗粒成分的杀菌作用敏感；（ii）两者对中性丝氨酸蛋白酶的杀菌作用均敏感；（iii）含组织蛋白酶G的组分对伴放线放线杆菌的杀伤作用与氧气和中性丝氨酸蛋白酶活性无关。
