Witte P L, Frantsve L M, Hergott M, Rahbe S M
Loyola University Medical Center, Maywood, IL 60153.
Eur J Immunol. 1993 Aug;23(8):1809-17. doi: 10.1002/eji.1830230812.
Many reports document that bone marrow stromal cells or their cytokine products can influence the formation of B cells in vitro. Most of this data comes from studies using lines or clones of stromal cells after multiple passage in culture, which could alter gene expression. Our aim in the present study was to determine which cytokines are produced by normal stromal cells under conditions that promote B lymphopoiesis. Primary cultured stromal cells were isolated on FACS from active Whitlock cultures. These cells proved to be relatively homogeneous in expression of cell surface antigens (CD44, VCAM-1, MECA10, and a molecule marked by hamster anti-mouse 8.28 monoclonal antibody). RNA from unselected Whitlock cultured adherent cells and sorted stromal cells from the same cultures were subjected to reverse transcriptase polymerase chain reaction to assess constitutive expression of several cytokine genes. Transcripts for interleukin-1 beta (IL-1 beta), IL-7, macrophage (M)-colony-stimulating factor (CSF), stem cell growth factor (SCGF), insulin-like growth factor 1 (IGF-1) and occasionally leukemia inhibitory factor were detected in RNA from intact cultures. Messages for IL-7, M-CSF, and SCGF were selectively contained within the isolated stromal cell fraction; whereas, IL-1 beta was found solely within the non-stromal cell fraction. IGF-1 was transcribed by both stromal cells and macrophages in Whitlock cultures. No evidence was found for constitutive expression of IL-1 alpha, IL-4, IL-6, or granulocyte-macrophage-CSF. This is in contrast to some reported stromal cell lines and clones. To determine if all primary stromal cells from active lymphopoietic cultures produced IL-7, the isolated cells were stained to reveal cytoplasmic IL-7 protein. A majority of the cells produced IL-7, but about 20% had no detectable IL-7 protein. Taken together, our results suggest that the primary stromal cells are a distinguishable cell type but functional subsets may exist. In regard to the differences in IL-7 production, the primary cell phenotype appears to mirror at least one division noted among the stromal cell lines.
许多报告记载，骨髓基质细胞或其细胞因子产物可在体外影响B细胞的形成。这些数据大多来自对培养中多次传代后的基质细胞系或克隆进行的研究，而传代可能会改变基因表达。我们在本研究中的目的是确定在促进B淋巴细胞生成的条件下，正常基质细胞会产生哪些细胞因子。从活跃的惠特洛克培养物中通过荧光激活细胞分选术（FACS）分离出原代培养的基质细胞。这些细胞在细胞表面抗原（CD44、血管细胞黏附分子-1、MECA10以及由仓鼠抗小鼠8.28单克隆抗体标记的一种分子）的表达上相对均一。对未分选的惠特洛克培养贴壁细胞以及来自同一培养物的分选基质细胞的RNA进行逆转录聚合酶链反应，以评估几种细胞因子基因的组成性表达。在完整培养物的RNA中检测到白细胞介素-1β（IL-1β）、IL-7、巨噬细胞（M）-集落刺激因子（CSF）、干细胞生长因子（SCGF）、胰岛素样生长因子1（IGF-1）的转录本，偶尔还检测到白血病抑制因子。IL-7、M-CSF和SCGF的信息选择性地存在于分离的基质细胞部分中；而IL-1β仅存在于非基质细胞部分中。在惠特洛克培养物中，IGF-1由基质细胞和巨噬细胞转录。未发现IL-1α、IL-4、IL-6或粒细胞-巨噬细胞-CSF组成性表达的证据。这与一些报道的基质细胞系和克隆不同。为了确定来自活跃淋巴细胞生成培养物的所有原代基质细胞是否都产生IL-7，对分离的细胞进行染色以显示细胞质IL-7蛋白。大多数细胞产生IL-7，但约20%的细胞未检测到IL-7蛋白。综上所述，我们的结果表明原代基质细胞是一种可区分的细胞类型，但可能存在功能亚群。关于IL-7产生的差异，原代细胞表型似乎反映了基质细胞系中至少一种已观察到的分化情况。
