Clohisy D R, Chappel J C, Teitelbaum S L
Department of Pathology and Laboratory Medicine, Jewish Hospital at Washington University Medical Center, St. Louis, Missouri 63110.
J Biol Chem. 1989 Apr 5;264(10):5370-7.
This study extends our previous observation that surface mannose receptor expression by pure populations of CSF-1-dependent bone marrow-derived macrophages increases with time (Clohisy, D. R., Bar-Shavit, Z., Chappel, J. C., and Teitelbaum, S. L. (1987) J. Biol. Chem. 262, 15922-15929). We presently find, however, that the progressive enhancement of 125I-mannose-bovine serum albumin (125I-Man-BSA) binding per cell reflects cell number rather than duration of culture. In fact, macrophages plated at high density bind 8-fold more 125I-Man-BSA than do their low density counterparts, with no difference in receptor-ligand affinity. Furthermore, cells cultured at high density are ultimately subjected to lower levels of exogenously provided macrophage growth factor, and fewer are in interphase. By obtaining synchronous populations of quiescent bone marrow macrophages, however, we demonstrate that neither cell cycling nor attendant levels of colony stimulating factor-1 influence mannose receptor expression. Our next series of experiments established that density-related mannose receptor expression reflects removal, by marrow macrophages, of a "down-regulating" factor contained in culture medium. To this end, we treated mononuclear phagocytes with either macrophage- or control-conditioned medium and found that, via a fetal calf serum-residing protein(s), only control medium is capable of noncompetitively reducing 125I-Man-BSA binding in a dose-dependent manner. Moreover, reconstituted 20-40% (NH4)2SO4-precipitable fractions derived from either sham-conditioned medium or fetal calf serum are capable of down-regulating mannose receptor expression. Alternatively, the same fraction obtained from macrophage-conditioned medium contains no such activity. Finally, initial characterization of the down-regulating factor reveals it to be acid-activable and trypsin-sensitive, yet resistant to heating to at least 80 degrees C, ribonuclease A, or freezing and thawing. We conclude that bone marrow macrophages up-regulate expression of their own plasma membrane mannose receptor by inactivating a noncompetitive, serum-residing inhibitory protein(s).
本研究扩展了我们之前的观察结果，即CSF-1依赖性骨髓来源巨噬细胞的纯群体中表面甘露糖受体的表达随时间增加（克洛希西，D.R.，巴尔-沙维特，Z.，查佩尔，J.C.，和泰特尔鲍姆，S.L.（1987年）《生物化学杂志》262，15922 - 15929）。然而，我们目前发现，每个细胞125I-甘露糖-牛血清白蛋白（125I-Man-BSA）结合的逐渐增强反映的是细胞数量而非培养持续时间。事实上，高密度接种的巨噬细胞比低密度接种的巨噬细胞结合的125I-Man-BSA多8倍，受体-配体亲和力没有差异。此外，高密度培养的细胞最终受到的外源性巨噬细胞生长因子水平较低，处于间期的细胞较少。然而，通过获得静止骨髓巨噬细胞的同步群体，我们证明细胞周期和集落刺激因子-1的伴随水平均不影响甘露糖受体的表达。我们接下来的一系列实验表明，与密度相关的甘露糖受体表达反映了骨髓巨噬细胞对培养基中一种“下调”因子的清除。为此，我们用巨噬细胞条件培养基或对照条件培养基处理单核吞噬细胞，发现仅对照培养基能够通过一种存在于胎牛血清中的蛋白质以剂量依赖性方式非竞争性地降低125I-Man-BSA的结合。此外，从假条件培养基或胎牛血清中重构的20 - 40%硫酸铵沉淀级分能够下调甘露糖受体的表达。相反，从巨噬细胞条件培养基中获得的相同级分不具有这种活性。最后，对下调因子的初步表征显示它对酸可激活且对胰蛋白酶敏感，但至少在80℃加热、核糖核酸酶A处理或冻融后仍具有抗性。我们得出结论，骨髓巨噬细胞通过使一种非竞争性的、存在于血清中的抑制蛋白失活来上调其自身质膜甘露糖受体的表达。
