Lemanske R F, Esser B, Kopp D, Asperheim M, Junk P, Tashoff T
J Allergy Clin Immunol. 1987 Jan;79(1):32-9. doi: 10.1016/s0091-6749(87)80013-1.
Intradermal injections of isolated mast cell granules (MCGs), as well as solubilized high-molecular-weight (HMW) (greater than 10,000 daltons) and low-molecular-weight (LMW) (10,000 greater than MW greater than 500 daltons) fractionated granule constituents, can produce inflammatory responses termed late-phase reactions (LPRs). The identity and mechanism of action of various inflammatory factor(s) contained within these fractions is incompletely established. Since rat LPRs are neutrophil-dependent responses, we analyzed the inherent neutrophil chemoattractant potential of HMW and LMW granule fractions using a 48-well microchemotaxis chamber. Although both HMW and LMW fractions attracted rat neutrophils, the LMW fraction was less active at equivalent protein concentrations. Checkerboard analysis demonstrated that the HMW fraction enhanced random migration of neutrophils, indicating that the HMW fraction contains factors that are primarily chemokinetic. To analyze further the HMW fraction, solubilized MCGs were sequentially fractionated with XM300 (MW greater than 300,000 daltons), and YM100 (300,000 greater than MW greater than 100,000 daltons), XM50 (100,000 greater than MW greater than 50,000 daltons), and YM10 (50,000 greater than MW greater than 10,000 daltons) ultrafiltration membranes. This process revealed that most in vivo inflammation-provoking activity as well as the in vitro chemoattractant activity resided in the XM300 and YM100 retentate fractions. Two of the major constituents of the HMW fraction, heparin and chymase, were evaluated for their contribution to the chemoattraction. Purified MCG heparin did not evoke neutrophil migratory responses in vitro or in vivo. Sepharose 4B chromatography of solubilized MCG demonstrated a peak of inflammation-provoking activity beginning at the void volume and tapering off near the 400,000 MW range. This in vivo activity was clearly separable from the chymase activity and represents the HMW inflammatory factors. These results demonstrate that both HMW and LMW granule fractions contain inflammatory activities capable of producing LPR in vivo and suggest that enhancement of neutrophil migration at sites of mast cell degranulation is one mechanism of action.
皮内注射分离出的肥大细胞颗粒（MCGs），以及溶解的高分子量（HMW）（大于10,000道尔顿）和低分子量（LMW）（10,000大于分子量大于500道尔顿）分级颗粒成分，可产生称为迟发性反应（LPRs）的炎症反应。这些分级成分中所含各种炎症因子的身份和作用机制尚未完全明确。由于大鼠LPRs是中性粒细胞依赖性反应，我们使用48孔微量趋化性小室分析了HMW和LMW颗粒分级成分固有的中性粒细胞趋化活性。尽管HMW和LMW分级成分均能吸引大鼠中性粒细胞，但在同等蛋白质浓度下，LMW分级成分的活性较低。棋盘分析表明，HMW分级成分增强了中性粒细胞的随机迁移，这表明HMW分级成分含有主要起化学促动作用的因子。为了进一步分析HMW分级成分，将溶解的MCGs依次用XM300（分子量大于300,000道尔顿）、YM100（300,000大于分子量大于100,000道尔顿）、XM50（100,000大于分子量大于50,000道尔顿）和YM10（50,000大于分子量大于10,000道尔顿）超滤膜进行分级。这一过程表明，大多数体内促炎活性以及体外趋化活性存在于XM300和YM100截留分级成分中。对HMW分级成分的两种主要成分肝素和糜酶对趋化作用的贡献进行了评估。纯化的MCG肝素在体外或体内均未引起中性粒细胞迁移反应。对溶解的MCG进行琼脂糖4B层析，结果显示在空体积处开始出现一个促炎活性峰，并在400,000分子量范围内逐渐减弱。这种体内活性与糜酶活性明显可分离，代表了HMW炎症因子。这些结果表明，HMW和LMW颗粒分级成分均含有能够在体内产生LPR的炎症活性，并提示肥大细胞脱颗粒部位中性粒细胞迁移的增强是一种作用机制。
