Kolotila M P, Diamond R D
Evans Memorial Department of Clinical Research, University Hospital, Boston University Medical Center, Massachusetts 02118.
Infect Immun. 1988 Aug;56(8):2016-22. doi: 10.1128/iai.56.8.2016-2022.1988.
We previously showed that unopsonized Candida albicans hyphae stimulated a delayed rise in the putative neutrophil second messengers Ca2+ and inositol 1,4,5-trisphosphate and subsequent O2- release, as compared with opsonized hyphae or zymosan. Therefore, cytoskeletal and degranulation temporal responses to these stimuli were examined. Unopsonized zymosan elicited no neutrophil responses under the experimental condition used. Neutrophil actin polymerization (quantitated by fluorescent measurements of NBD phallacidin) was rapid after stimulation by opsonized hyphae or zymosan (peaking at 1 and 2 min, respectively). This corresponded to observed changes in microscopic actin polymerization, measured with rhodamine phalloidin, which progressed from initially diffuse to collarlike to cylinderlike staining patterns surrounding the hyphae. Compared with opsonized hyphae, unopsonized hyphae resulted in a delayed appearance of the last two visible patterns (P less than 0.05) and in quantitative actin polymerization despite similarly rapid initial contact and spreading over the hyphae by neutrophils. Unlike other neutrophil responses, degranulation did not follow the delayed patterns of responses to stimulation with unopsonized hyphae. In the absence of the release of the cytoplasmic marker lactate dehydrogenase, the release of beta-glucuronidase, an azurophil granule marker, gradually and progressively rose in response to all of the stimuli but unopsonized zymosan. The low but significant levels observed were within a range consistent with published results for degranulation responses to particulate stimuli without cytochalasin B. A quantitative immunoassay of lactoferrin, a specific granule marker, detected no release into supernatants, and immunofluorescent staining indicated concomitant depletion of lactoferrin from neutrophil granules and binding to hyphal and neutrophil surfaces after stimulation by unopsonized hyphae. Thus, the delayed actin polymerization response to unopsonized hyphae occurred subsequent to neutrophil attachment and spreading and resembled the temporal sequence of other neutrophil responses linked to the respiratory burst. In contrast, the degranulation responses to all stimuli appeared to begin and progress gradually after observed attachment and spreading of the neutrophil over hyphal surfaces without a clear temporal relationship to rises in cytoplasmic Ca2+ or F-actin. In addition, the avid binding of released lactoferrin to cell surfaces eliminates its value as a quantitative marker of enzyme release but raises the possibility that it might participate in fungicidal activity.(ABSTRACT TRUNCATED AT 400 WORDS)
我们之前的研究表明，与经调理素作用的菌丝或酵母聚糖相比，未经调理素作用的白色念珠菌菌丝可刺激假定的中性粒细胞第二信使Ca2+和肌醇1,4,5 -三磷酸出现延迟升高，并随后释放O2-。因此，我们检测了细胞骨架和脱颗粒对这些刺激的时间反应。在所用实验条件下，未经调理素作用的酵母聚糖未引发中性粒细胞反应。经调理素作用的菌丝或酵母聚糖刺激后，中性粒细胞肌动蛋白聚合（通过NBD鬼笔环肽荧光测量定量）迅速发生（分别在1分钟和2分钟达到峰值）。这与用罗丹明鬼笔环肽测量的显微镜下肌动蛋白聚合变化相对应，其从最初的弥散状发展为菌丝周围的环状再到圆柱状染色模式。与经调理素作用的菌丝相比，未经调理素作用的菌丝导致后两种可见模式出现延迟（P小于0.05），且肌动蛋白定量聚合，尽管中性粒细胞与菌丝的初始接触和铺展同样迅速。与其他中性粒细胞反应不同，脱颗粒并未遵循对未经调理素作用的菌丝刺激的延迟反应模式。在无细胞质标记物乳酸脱氢酶释放的情况下，嗜天青颗粒标记物β -葡萄糖醛酸酶的释放对所有刺激（除未经调理素作用的酵母聚糖外）均逐渐升高。观察到的低但显著的水平在与已发表的无细胞松弛素B时颗粒刺激脱颗粒反应结果一致的范围内。乳铁蛋白（一种特异性颗粒标记物）的定量免疫测定未检测到其释放到上清液中，免疫荧光染色表明经未经调理素作用的菌丝刺激后，乳铁蛋白从中性粒细胞颗粒中同时耗尽并结合到菌丝和中性粒细胞表面。因此，对未经调理素作用的菌丝的延迟肌动蛋白聚合反应发生在中性粒细胞附着和铺展之后，类似于与呼吸爆发相关的其他中性粒细胞反应的时间顺序。相比之下，对所有刺激的脱颗粒反应似乎在观察到中性粒细胞在菌丝表面附着和铺展后开始并逐渐进行，与细胞质Ca2+或F -肌动蛋白的升高无明确的时间关系。此外，释放的乳铁蛋白与细胞表面的 avid 结合消除了其作为酶释放定量标记物的价值，但增加了其可能参与杀真菌活性的可能性。（摘要截断于400字）
