Smail E H, Cronstein B N, Meshulam T, Esposito A L, Ruggeri R W, Diamond R D
Evans Memorial Department of Clinical Research, University Hospital, Boston University Medical Center, MA 02118.
J Immunol. 1992 Jun 1;148(11):3588-95.
We previously described a lyophilized supernatant from germinated Candida albicans that blocks human neutrophil (PMN) O2- production and degranulation stimulated by several PMN agonists but does not block stimulation by PMA. In studies to further characterize this Candida hyphal inhibitory product (CHIP), we noted several physicochemical parallels with the purine nucleoside adenosine (Ado). A Sephadex G-10 semipurified fraction of CHIP had an absorption peak near 260 nm, an apparent m.w. of less than 400, and was resistant to boiling and proteases. Maximally effective doses of CHIP (100 micrograms/ml) and Ado (100 microM) blocked 0.1 microM FMLP-stimulated O2- production by 76.8 +/- 4.1 and 81.7 +/- 4.8%, respectively. Ado deaminase, known to inactivate Ado, reversed inhibition by both Ado and CHIP. Results were comparable for the effect of CHIP and Ado on FMLP-stimulated beta-glucuronidase and lactoferrin release. Activation of the respiratory burst by opsonized C. albicans yeast was also inhibited by CHIP and Ado, but the extent of inhibition was less than for FMLP. At yeast:PMN ratios of 4:1, 10:1, and 40:1, CHIP inhibited O2- by -3.8%, 14.3%, and 12.8%, respectively; Ado blocked production by 32.9%, 24.2%, and 11.5%, respectively. The effect of CHIP and Ado on Candida killing by PMN was compared using two viability assays in each of four experiments. Ado (100 microM) had no effect on killing, although CHIP (100 micrograms/ml) inhibited killing in the MTT assay at 15 and 45 min by 81.6 +/- 6.3 and 24.7 +/- 6.2%, respectively; as assayed by CFU, CHIP inhibited killing by 34.1 +/- 6.2 and 10.3 +/- 2.5%, respectively. The ability of CHIP to inhibit killing was not affected by adding Ado deaminase, providing additional evidence that an Ado-like effect by CHIP is not essential for killing inhibition. Killing of opsonized Streptococcus pneumoniae was also inhibited in a concentration-dependent manner. Reverse-phase HPLC of the semipurified fraction revealed a peak, eluting identically to authentic Ado, which was eliminated by adding Ado deaminase. Ado content of the G-10 fraction was sufficient to account fully for the FMLP-inhibitory activity. The antikilling activity was resistant to boiling and proteases but was eliminated by mild periodation. Fractions eluting from a Sephadex CL6B column between 0.8 and 2.0 x 10(6) m.w. had increased sp. act. for killing inhibition. Sp. act. increased as carbohydrate content increased, but killing inhibition by various Candida cell wall constituents was absent to modest compared to inhibition induced by CHIP.(ABSTRACT TRUNCATED AT 400 WORDS)
我们之前描述过一种来自白色念珠菌芽管的冻干上清液，它能阻断人中性粒细胞（PMN）的O₂⁻生成以及几种PMN激动剂刺激引起的脱颗粒，但不阻断佛波酯（PMA）的刺激。在进一步表征这种念珠菌菌丝抑制产物（CHIP）的研究中，我们注意到它与嘌呤核苷腺苷（Ado）在一些物理化学性质上有相似之处。CHIP经葡聚糖G - 10半纯化后的组分在260nm附近有一个吸收峰，表观分子量小于400，且对煮沸和蛋白酶具有抗性。CHIP的最大有效剂量（100μg/ml）和Ado（100μM）分别使0.1μM 甲酰甲硫氨酸 - 亮氨酸 - 苯丙氨酸（FMLP）刺激的O₂⁻生成减少76.8±4.1%和81.7±4.8%。已知能使Ado失活的腺苷脱氨酶可逆转Ado和CHIP的抑制作用。CHIP和Ado对FMLP刺激的β - 葡萄糖醛酸酶和乳铁蛋白释放的影响结果相似。调理后的白色念珠菌酵母激活呼吸爆发也受到CHIP和Ado的抑制，但抑制程度小于对FMLP的抑制。在酵母与PMN比例为4:1、10:1和40:1时，CHIP分别使O₂⁻生成减少3.8%、14.3%和12.8%；Ado分别使生成减少32.9%、24.2%和11.5%。在四个实验中的每个实验都使用两种活力测定方法比较了CHIP和Ado对PMN杀灭念珠菌的影响。Ado（100μM）对杀灭无影响，而CHIP（100μg/ml）在MTT测定中分别在15分钟和45分钟时使杀灭减少81.6±6.3%和24.7±6.2%；通过菌落形成单位（CFU）测定，CHIP分别使杀灭减少34.1±6.2%和10.3±2.5%。添加腺苷脱氨酶不影响CHIP抑制杀灭的能力，这提供了额外证据表明CHIP类似Ado的作用对于抑制杀灭并非必需。调理后的肺炎链球菌的杀灭也以浓度依赖方式受到抑制。半纯化组分的反相高效液相色谱显示一个峰，其洗脱情况与纯品Ado相同，添加腺苷脱氨酶可消除该峰。G - 10组分中的Ado含量足以完全解释其对FMLP的抑制活性。抗杀灭活性对煮沸和蛋白酶具有抗性，但可被温和的高碘酸盐处理消除。从葡聚糖CL6B柱上洗脱的分子量在0.8至2.0×10⁶之间的组分对杀灭抑制的比活性增加。比活性随碳水化合物含量增加而增加，但与CHIP诱导的抑制相比，各种念珠菌细胞壁成分对杀灭的抑制作用不明显或较弱。（摘要截断于400字）
