IDENTIFICATION OF CYSTATIN-C, A CYSTEINE PROTEINASE-INHIBITOR, AS A MAJOR SECRETORY PRODUCT OF HUMAN ALVEOLAR MACROPHAGES INVITRO
IDENTIFICATION OF CYSTATIN-C, A CYSTEINE PROTEINASE-INHIBITOR, AS A MAJOR SECRETORY PRODUCT OF HUMAN ALVEOLAR MACROPHAGES INVITRO
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DOI：
10.1164/ajrccm/141.3.698
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发表时间：
1990-03-01
期刊：
影响因子：
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通讯作者：
GRUBB, A
中科院分区：
文献类型：
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作者：
CHAPMAN, HA;REILLY, JJ;GRUBB, A
半胱氨酸蛋白酶抑制剂C是在人体体液（如脊髓液、乳汁和精浆）中发现的半胱氨酸类蛋白酶的主要抑制剂。在这项研究中，我们表明，人支气管肺泡液也含有胱抑素C和检查胱抑素C的表达肺泡巨噬细胞在体外。代谢标记的细胞提取物的免疫沉淀和细胞提取物和培养基的免疫印迹显示，半胱氨酸蛋白酶抑制剂C是作为14（±）0.5)14千道尔顿（kD）蛋白质，并且大于90%的蛋白质作为14 kD产物释放到培养物上清液中（26.5 ± 0.5）。6.8 ng/106个细胞/24小时）。半胱氨酸蛋白酶抑制剂C是在体外最初24小时期间分泌的最丰富的蛋白质之一，代表约正常非吸烟者巨噬细胞释放的总蛋白质的10%至12%。从吸烟者或非吸烟者的巨噬细胞暴露于酵母多糖在体外获得的肺泡巨噬细胞释放的胱抑素C比非吸烟者的巨噬细胞少10%至55%。我们还检测了吸烟者和非吸烟者巨噬细胞培养上清液中的功能性胱抑素C。非吸烟者巨噬细胞的上清液抑制cathespin B样amidolytic活性在荧光测定在pH 5.5。通过用Sepharose偶联的胱抑素C抗体吸附来阻断抑制作用，随后从Sepharose珠中回收抑制剂。相反，吸烟者巨噬细胞的上清液具有明显的cathespin B样活性。用固相胱抑素C抗体吸附吸烟者细胞上清液后，组织蛋白酶B样活性增加超过100%，证实了免疫印迹数据，表明吸烟者巨噬细胞也释放胱抑素C。这些数据表明，半胱氨酸蛋白酶抑制剂C是肺泡巨噬细胞的主要组成性分泌产物，通常掩盖这些细胞分泌的半胱氨酸蛋白酶的活性。在炎症过程中，如吸烟引起的炎症过程中，胱抑素C的释放下调，导致巨噬细胞微环境中酸性蛋白酶活性增加。这种酶和抑制剂释放平衡的改变可能有助于巨噬细胞介导的结缔组织重塑，已知这是炎症过程的一部分。
The major inhibitor of the cysteine class of proteinases found in human body fluids, such as spinal fluid, milk, and seminal plasma, is cystatin C. In this study we show that human bronchoalveolar fluid also contains cystatin C and examine cystatin C expression by alveolar macrophages in vitro. Immunoprecipitation of extracts of metabolically labeled cells and immunoblotting of cellular extracts and culture media show that cystatin C is synthesized as a 14 (.+-. 0.5) kilodalton (kD) protein and that greater than 90% of the protein is released as the 14 kD product into the culture supernatant (26.5 .+-. 6.8 ng per 106 cells per 24 h). Cystatin C is one of the most abundant proteins secreted during the first 24 h in vitro, representing .apprx. 10 to 12% of the total protein released by normal nonsmoker macrophages. Alveolar macrophages obtained from cigarette smokers or nonsmoker macrophages exposed to zymosan in vitro released 10 to 55% less cystatin C than nonsmoker macrophages. We also assayed culture supernatants from macrophages of smokers and nonsmokers for functional cystatin C. Supernatants of nonsmoker macrophages inhibited cathespin B-like amidolytic activity in a fluorometric assay at pH 5.5. The inhibition was blocked by adsorption with Sepharose-coupled cystatin C antibodies and the inhibitor subsequently recovered from the Sepharose beads. In contrast, supernatants from smoker macrophages had obvious cathespin B-like activity. Following adsorption of smoker cell supernatants with the solid-phase cystatin c antibodies, cathepsin B-like activity increased over 100%, confirming the immunoblot data indicating that smoker macrophages also release cystatin C. These data indicate that cystatin C is a major constitutive secretory product of alveolar macrophages and normally masks the activity of cysteine proteinases also secreted by these cells. During inflammatory processes, such as that produced by cigarette smoking, cystatin C release is downregulated, contributing to increased acidic proteinase activity in the macrophage microenvironment. This altered balance of enzyme and inhibitor release may contribute to the macrophage-mediated connective tissue remodeling known to be a part of inflammatory processes.