O'Shea J J, Brown E J, Seligmann B E, Metcalf J A, Frank M M, Gallin J I
J Immunol. 1985 Apr;134(4):2580-7.
In this report, the modulation and localization of complement receptors CR1 and CR3 in neutrophils were examined with the use of monoclonal antibodies (mab) directed against these membrane proteins. We first studied complement receptor modulation in a patient with neutrophil-specific granule deficiency. With flow cytometric analysis, we determined that, while N-formyl-methionyl-leucyl-phenylalanine (f-met-leu-phe) (10(-6) M) caused an increase in the binding of both anti-CR1 and anti-CR3 mab to normal neutrophils, the fmet-leu-phe-stimulated neutrophils from our patient increased anti-CR1 binding but decreased anti-CR3 binding. This suggested that CR3, but not CR1, might be associated with specific granules. We next studied receptor modulation in organelle-depleted neutrophil cytoplasts obtained from normal donors. Unlike the specific granule-deficient neutrophils, the normal cytoplasts failed to augment expression of either receptor after stimulation. Immunofluorescence studies of permeabilized polymorphonuclear leukocytes (PMN) revealed considerable internal binding of both anti-CR1 and anti-CR3. In additional studies, phorbol myristate acetate (PMA) was used as a stimulus for receptor modulation in normal neutrophils. Unlike fmet-leu-phe and C5a, PMA elicited a biphasic dose-response curve. High doses of PMA (greater than 0.5 ng/ml) caused a reduction in the magnitude of membrane expression of both CR1 and CR3. In studies designed to localize the internal pool of receptors, we evaluated the binding of 125I-anti-receptor mab to plasma membrane-, specific granule, and azurophilic granule-enriched fractions obtained from sucrose gradient fractionation of disrupted neutrophils. 125I-anti-CR1 mab bound to the membrane-enriched fraction but bound little to either granule-enriched fraction. In contrast, 125I-anti-CR3 mab bound more to the specific granule-enriched fraction than to the plasma membrane-enriched fraction. Azurophilic granules showed no increased anti-CR3 binding. Immunoprecipitation of radiolabeled solubilized subcellular fractions with anti-receptor mab confirmed these findings. CR3 was present in the plasma membrane-, and specific granule-enriched fraction but not in the azurophilic granule-enriched fraction. CR1, however, was present only in the plasma membrane-enriched fraction. These data indicate that there are intracellular pools for both the CR1 and CR3, but the intracellular locations for these pools are distinct. The pool for CR3 co-sediments with specific granules, while the pool for CR1 does not. Nonetheless, a variety of stimulatory agents increase and decrease the membrane expression of both receptors in parallel.
在本报告中，我们使用针对这些膜蛋白的单克隆抗体（mab）研究了中性粒细胞中补体受体CR1和CR3的调节与定位。我们首先研究了一名患有中性粒细胞特异性颗粒缺乏症患者的补体受体调节情况。通过流式细胞术分析，我们确定，虽然N-甲酰甲硫氨酰-亮氨酰-苯丙氨酸（f-met-leu-phe）（10⁻⁶ M）可使抗CR1和抗CR3 mab与正常中性粒细胞的结合增加，但来自我们患者的经fmet-leu-phe刺激的中性粒细胞增加了抗CR1结合，但降低了抗CR3结合。这表明CR3而非CR1可能与特异性颗粒相关。接下来，我们研究了从正常供体获得的细胞器缺失的中性粒细胞胞质体中的受体调节情况。与特异性颗粒缺乏的中性粒细胞不同，正常胞质体在刺激后未能增强任何一种受体的表达。对透化的多形核白细胞（PMN）的免疫荧光研究显示，抗CR1和抗CR3均有大量的内部结合。在其他研究中，佛波酯肉豆蔻酸酯乙酸酯（PMA）被用作正常中性粒细胞受体调节的刺激物。与fmet-leu-phe和C5a不同，PMA引发了双相剂量反应曲线。高剂量的PMA（大于0.5 ng/ml）导致CR1和CR3的膜表达量均降低。在旨在定位受体内部池的研究中，我们评估了¹²⁵I-抗受体mab与从破碎的中性粒细胞蔗糖梯度分级分离获得的富含质膜、特异性颗粒和嗜天青颗粒的级分的结合情况。¹²⁵I-抗CR1 mab与富含膜的级分结合，但与富含颗粒的级分结合很少。相比之下，¹²⁵I-抗CR3 mab与富含特异性颗粒的级分的结合多于与富含质膜的级分的结合。嗜天青颗粒未显示抗CR3结合增加。用抗受体mab对放射性标记的可溶性亚细胞级分进行免疫沉淀证实了这些发现。CR3存在于质膜和富含特异性颗粒的级分中，但不存在于富含嗜天青颗粒的级分中。然而，CR1仅存在于富含质膜的级分中。这些数据表明，CR1和CR3均有细胞内池，但这些池的细胞内位置不同。CR3的池与特异性颗粒共沉降，而CR1的池则不然。尽管如此，多种刺激剂会同时增加和降低两种受体的膜表达。
