Landreth K S, Dorshkind K
Department of Microbiology, West Virginia University, Medical Center, Morgantown 26506.
J Immunol. 1988 Feb 1;140(3):845-52.
Two bone marrow stromal cell lines isolated from the adherent layer of a Dexter-type long term bone marrow culture differ markedly in their hemopoietic support capacity. S17 supports myelopoiesis and the differentiation of early B cell precursors into B lymphocytes while S10 supports myeloid cell differentiation and not B lymphopoiesis. The identification of a stromal cell line with B cell support capacity prompted an investigation of whether the effects of S17 were mediated via soluble factors. Results presented herein indicate that medium conditioned by S17 but not S10 contains an activity that can induce the expression of the 220,000 m.w. 14.8 antigen and cytoplasmic mu H chain of Ig in B lymphocyte progenitors that have not yet expressed these markers. Bone marrow cells were depleted of 14.8+, cytoplasmic mu+ pre-B cells on antibody-coated petri dishes. After 24-h liquid culture newly generated pre-B cells were enumerated as cells that expressed cytoplasmic mu H chain of Ig but not Ig L chains by immunofluorescence. Expression of Ly5(220) was monitored by 14.8 antibody binding. This pre-B cell differentiation activity was abrogated by digestion with pronase, aminopeptidase, or carboxypeptidase. Isoelectric focusing data revealed the activity to have isoelectric point of 5.9 to 6.2. S17-conditioned medium was fractionated using HPLC and each fraction tested for pre-B cell-generating activity. Fractions collected from a Superose 12 gel filtration column were found to have two peaks of activity associated with molecules of apparent m.w. of approximately 60,000 and 10,000. Virtually identical peaks of activity were observed when medium conditioned by heterogeneous stromal cell cultures was fractionated. Separation of S10-conditioned medium revealed no cryptic activity. S17-conditioned medium was further characterized by anion exchange chromatography and the majority of the pre-B cell generating activity shown to be associated with the void volume that eluted from a MonoQ column. These fractions were rechromatographed on Superose and the activity again found to be associated with two fractions corresponding to apparent m.w. of 60,000 and 10,000. The S17 pre-B cell differentiation activity appears to result from the presence of a novel molecule because other well characterized mediators had no activity in this short-term liquid culture system. No pre-B cell-generating activity was observed when IL-1 or conditioned medium containing IL-2, IL-3, or IL-4 (B cell stimulatory factor 1) were added to cultures.(ABSTRACT TRUNCATED AT 400 WORDS)
从德克斯特型长期骨髓培养物的贴壁层分离出的两种骨髓基质细胞系，其造血支持能力有显著差异。S17支持髓细胞生成以及早期B细胞前体向B淋巴细胞的分化，而S10支持髓样细胞分化但不支持B淋巴细胞生成。具有B细胞支持能力的基质细胞系的鉴定促使人们研究S17的作用是否通过可溶性因子介导。本文给出的结果表明，由S17而非S10条件培养的培养基含有一种活性物质，它能在尚未表达这些标志物的B淋巴细胞祖细胞中诱导220,000分子量的14.8抗原和Ig的细胞质μ重链的表达。在包被抗体的培养皿上，骨髓细胞中的14.8 +、细胞质μ + 前B细胞被清除。经过24小时液体培养后，新产生的前B细胞通过免疫荧光法被计为表达Ig的细胞质μ重链但不表达Ig轻链的细胞。Ly5(220)的表达通过14.8抗体结合来监测。这种前B细胞分化活性在用链霉蛋白酶、氨肽酶或羧肽酶消化后被消除。等电聚焦数据显示该活性的等电点为5.9至6.2。使用高效液相色谱法对S17条件培养的培养基进行分级分离，并对每个级分进行前B细胞生成活性测试。从Superose 12凝胶过滤柱收集的级分被发现有两个活性峰，与表观分子量约为60,000和10,000的分子相关。当对由异质性基质细胞培养条件培养的培养基进行分级分离时，观察到几乎相同的活性峰。对S10条件培养的培养基进行分离未发现隐匿活性。通过阴离子交换色谱法对S17条件培养的培养基进行进一步表征，结果表明大部分前B细胞生成活性与从MonoQ柱洗脱的空体积相关。这些级分在Superose上再次进行色谱分离，活性再次被发现与两个级分相关，其表观分子量分别为60,000和10,000。S17的前B细胞分化活性似乎源于一种新分子的存在，因为其他特征明确的介质在这个短期液体培养系统中没有活性。当将IL - 1或含有IL - 2、IL - 3或IL - 4（B细胞刺激因子1）的条件培养基添加到培养物中时，未观察到前B细胞生成活性。（摘要截断于400字）
