MacGlashan D, Botana L M
Johns Hopkins University, Asthma and Allergy Center, Baltimore, MD 21224.
J Immunol. 1993 Feb 1;150(3):980-91.
Human basophils exhibited biphasic cytosolic Ca2+ responses after stimulation with both the bacterial peptide, FMLP and IgE cross-linking agents such as goat polyclonal anti-IgE PS myeloma (anti-IgE) antibody and Ag. The biphasic response to anti-IgE antibody was not apparent unless supraoptimal concentrations of anti-IgE antibody were used to stimulate the cells. Biphasic response characteristics were also observed in single cells. As in many cell types, the first phase of the Ca2+ response appeared independent of extracellular Ca2+. Challenging basophils with anti-IgE in the presence of EGTA, added simultaneously or 3 to 5 min before the stimulus, had little effect on the first transient Ca2+ elevation while completely eliminating the second phase of the response. We noted that while simultaneous addition of EGTA had little effect on the histamine release after challenge with FMLP, there was no histamine release after the simultaneous addition of anti-IgE and EGTA. An examination of the kinetics of histamine release after anti-IgE also demonstrated that no histamine release occurs during the initial transient response. Since the initial Ca2+ transient was intact after both stimuli in the presence of EGTA, we concluded that the initial transient was not sufficient to induce histamine release. The hypothesis that distinct sources of calcium were responsible for the initial Ca2+ transient that followed challenge with FMLP peptide or anti-IgE antibody was also tested. We found that prior treatment of basophils with thapsigargin inhibited the initial Ca2+ response that followed both FMLP and anti-IgE antibody. Furthermore, stimulation with FMLP, in the presence of EGTA, depleted the calcium source responsible for the anti-IgE response. The experiment conducted in reverse order showed that anti-IgE antibody could discharge the calcium source responsible for the initial FMLP Ca2+ response. These experiments indicated that FMLP and anti-IgE antibody use the same internal source of calcium to produce the initial Ca2+ response. Taken together with previous studies on FMLP, anaphylatoxin split product of the C component, C5; and platelet activating factor-induced release in human basophils, these results suggest that the initial transient elevation, which is relatively independent of extracellular Ca2+, is not sufficient to induce degranulation.
人类嗜碱性粒细胞在用细菌肽FMLP以及IgE交联剂（如山羊多克隆抗IgE PS骨髓瘤（抗IgE）抗体和抗原）刺激后，表现出双相性胞质Ca2+反应。除非使用超最佳浓度的抗IgE抗体来刺激细胞，否则对抗IgE抗体的双相反应并不明显。在单个细胞中也观察到了双相反应特征。与许多细胞类型一样，Ca2+反应的第一阶段似乎与细胞外Ca2+无关。在EGTA存在的情况下，在刺激前同时添加或提前3至5分钟添加EGTA来刺激嗜碱性粒细胞，对第一个短暂的Ca2+升高影响很小，而完全消除了反应的第二阶段。我们注意到，虽然同时添加EGTA对FMLP刺激后的组胺释放影响很小，但同时添加抗IgE和EGTA后则没有组胺释放。对抗IgE刺激后组胺释放动力学的研究还表明，在最初的短暂反应期间没有组胺释放。由于在EGTA存在的情况下，两种刺激后最初的Ca2+瞬变都是完整的，我们得出结论，最初的瞬变不足以诱导组胺释放。我们还测试了一种假设，即不同的钙来源负责FMLP肽或抗IgE抗体刺激后最初的Ca2+瞬变。我们发现，用毒胡萝卜素预先处理嗜碱性粒细胞会抑制FMLP和抗IgE抗体刺激后最初的Ca2+反应。此外，在EGTA存在的情况下用FMLP刺激会耗尽负责抗IgE反应的钙源。以相反顺序进行的实验表明，抗IgE抗体可以耗尽负责最初FMLP Ca2+反应的钙源。这些实验表明，FMLP和抗IgE抗体使用相同的内部钙源来产生最初的Ca2+反应。结合先前关于FMLP、补体C5的过敏毒素裂解产物以及血小板活化因子诱导人嗜碱性粒细胞释放的研究，这些结果表明，相对独立于细胞外Ca2+的最初短暂升高不足以诱导脱颗粒。
