O'Garra A, Stapleton G, Dhar V, Pearce M, Schumacher J, Rugo H, Barbis D, Stall A, Cupp J, Moore K
Department of Immunology, DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, CA 94304.
Int Immunol. 1990;2(9):821-32. doi: 10.1093/intimm/2.9.821.
We have examined a panel of murine Ly-1+ B lymphomas and purified normal murine peritoneal B cells separated into subsets on the basis of expression of the Ly-1 surface antigen, for their ability to produce cytokines. Where possible, we have used a combination of cytokine detection methods in order to compensate for differences in sensitivity and specificity, and the possibility of inhibitors masking an activity. All the lymphomas tested were shown to constitutively express TGF-beta and CSIF/IL-10. In addition, varying levels of IL-6, TNF-alpha and TNF-beta, and G-CSF, were demonstrable in most of the lymphomas, and variants of one lymphoma (CH12) additionally produced varying levels of IL-3, IL-4, and GM-CSF. FACS purified normal Ly-1+ and Ly-1- peritoneal B cells, were also shown to express RNA encoding CSIF/IL-10, IL-6, TNF-alpha and TNF-beta, and very low levels of G-CSF, following stimulation with LPS. These data were supported by the detection of IL-6 and CSIF/IL-10 in supernatants from LPS-stimulated Ly-1+ and Ly-1- B cells using specific immunoassays. None of the lymphomas or B cell preparations produced IL-1 alpha, IL-2, IL-5, IL-7, or IFN-gamma. The purity of our normal B cell populations was assessed by phenotypic analysis on the FACS and also by the disappearance of certain mRNA transcripts after purification, e.g. CD4, c-fms, GM-CSF, and IFN-gamma, most of which could be detected in LPS-stimulated total peritoneal cell populations. This suggested that our B cell purification method had reduced, to a level undetectable in our assays, contaminating T cells (CD4), macrophages (c-fms, GM-CSF), and NK cells (IFN-gamma). Absence of IL-3, IL-4, IL-5, and GM-CSF expression by LPS-stimulated Ly-1+ and Ly-1- B cells reduced the concern that contaminating peritoneal mast cells could account for the observed cytokine production. We therefore believe our data provide strong support for production of a subset of cytokines by LPS-stimulated normal B cells. Both the Ly-1+ B lymphomas and normal Ly-1+ and Ly-1- B cells appear capable of expressing IL-6, TNF-alpha, TNF-beta, and CSIF/IL-10.(ABSTRACT TRUNCATED AT 400 WORDS)
我们检测了一组小鼠Ly-1+B淋巴瘤以及根据Ly-1表面抗原表达分离成亚群的纯化正常小鼠腹腔B细胞产生细胞因子的能力。在可能的情况下，我们使用了多种细胞因子检测方法，以弥补灵敏度和特异性的差异，以及抑制剂掩盖活性的可能性。所有检测的淋巴瘤均显示组成性表达转化生长因子-β（TGF-β）和细胞因子合成抑制因子/白细胞介素-10（CSIF/IL-10）。此外，在大多数淋巴瘤中可检测到不同水平的白细胞介素-6（IL-6）、肿瘤坏死因子-α（TNF-α）、肿瘤坏死因子-β（TNF-β）和粒细胞集落刺激因子（G-CSF），一种淋巴瘤（CH12）的变体还产生了不同水平的白细胞介素-3（IL-3）、白细胞介素-4（IL-4）和粒细胞-巨噬细胞集落刺激因子（GM-CSF）。经荧光激活细胞分选（FACS）纯化的正常Ly-1+和Ly-1-腹腔B细胞，在脂多糖（LPS）刺激后也显示表达编码CSIF/IL-10、IL-6、TNF-α和TNF-β的RNA，以及极低水平的G-CSF。使用特异性免疫测定法在LPS刺激的Ly-1+和Ly-1- B细胞的上清液中检测到IL-6和CSIF/IL-10，支持了这些数据。没有一种淋巴瘤或B细胞制剂产生白细胞介素-1α（IL-1α）、白细胞介素-2（IL-2）、白细胞介素-5（IL-5）、白细胞介素-7（IL-7）或干扰素-γ（IFN-γ）。我们通过FACS上的表型分析以及纯化后某些mRNA转录本的消失来评估正常B细胞群体的纯度，例如CD4、c-fms、GM-CSF和IFN-γ，其中大多数可以在LPS刺激的总腹腔细胞群体中检测到。这表明我们的B细胞纯化方法已将污染的T细胞（CD4）、巨噬细胞（c-fms、GM-CSF）和自然杀伤细胞（IFN-γ）减少到我们的检测方法无法检测的水平。LPS刺激的Ly-1+和Ly-1- B细胞不表达IL-3、IL-4、IL-5和GM-CSF，这减少了对污染的腹腔肥大细胞可能导致观察到的细胞因子产生的担忧。因此，我们认为我们的数据为LPS刺激的正常B细胞产生细胞因子亚群提供了有力支持。Ly-1+B淋巴瘤以及正常Ly-1+和Ly-1- B细胞似乎都能够表达IL-6、TNF-α、TNF-β和CSIF/IL-10。（摘要截断于400字）
