Brossart P, Grünebach F, Stuhler G, Reichardt V L, Möhle R, Kanz L, Brugger W
University of Tübingen, Department of Hematology, Oncology and Immunology, Tübingen, Germany.
Blood. 1998 Dec 1;92(11):4238-47.
Recently it has been shown that dendritic cells (DC) can develop from peripheral blood monocytes when grown in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). However, it is unclear whether DC can also develop from monocytes in absence of these cytokines. We therefore analyzed the effect of Flt-3 ligand (Flt3L) and of CD40 ligand on the development of human DC from blood monocytes in the absence of GM-CSF. Adherent peripheral blood mononuclear cells (PBMNC) were cultured in the presence of different cytokine combinations and analyzed for the expression of surface molecules and antigen presenting capacity. For functional analyses, cells were tested for their ability to stimulate allogeneic T lymphocytes in a mixed lymphocyte reaction (MLR), to present soluble antigens, and to induce primary HIV-peptide-specific cytotoxic T-cell (CTL) responses in vitro. Furthermore, expression of DC-CK1, a recently identified chemokine with specific expression in DC, and of IL-18 (IGIF), a growth and differentiation factor for Th 1 lymphocytes, was analyzed by reverse-transcription polymerase chain reaction (RT-PCR). In our study, Flt3L alone was not sufficient to generate DC and required addition of IL-4. DC generated with Flt3L and IL-4 underwent maturation after stimulation with tumor necrosis factor- (TNF-) or CD40L, characterized by CD83 expression, upregulation of MHC, adhesion, and costimulatory molecules as well as increased allogeneic proliferative response. In contrast, CD40 ligation alone promoted differentiation of adherent blood monocytes into functional DC in the absence of GM-CSF and IL-4. These cells displayed all phenotypic and functional characteristics of mature DC and were potent stimulatory cells in priming of major histocompatibility complex (MHC) class I-restricted CTL responses against an HIV-peptide, whereas their ability to present soluble protein antigens was reduced. Using a semiquantitative RT-PCR, DC-CK1 and IL-18 transcripts were detected in all generated DC populations, independent of growth factors used. Our findings provide further evidence for the importance of CD40-CD40L interaction for initiation and maintenance of T-cell responses and confirm the emerging concept that blood monocytes provide an additional source of DC depending on external stimuli.
最近研究表明，在外周血单核细胞于粒细胞-巨噬细胞集落刺激因子（GM-CSF）和白细胞介素-4（IL-4）存在的情况下培养时，可发育为树突状细胞（DC）。然而，尚不清楚在缺乏这些细胞因子时DC是否也能从单核细胞发育而来。因此，我们分析了在缺乏GM-CSF的情况下，Flt-3配体（Flt3L）和CD40配体对人血单核细胞发育为DC的影响。将贴壁外周血单核细胞（PBMNC）在不同细胞因子组合存在的情况下进行培养，并分析其表面分子的表达和抗原呈递能力。为进行功能分析，检测细胞在混合淋巴细胞反应（MLR）中刺激同种异体T淋巴细胞、呈递可溶性抗原以及在体外诱导原发性HIV肽特异性细胞毒性T细胞（CTL）反应的能力。此外，通过逆转录聚合酶链反应（RT-PCR）分析了DC-CK1（一种最近鉴定出的在DC中特异性表达的趋化因子）和IL-18（IFN-γ诱导因子，Th1淋巴细胞的生长和分化因子）的表达。在我们的研究中，单独的Flt3L不足以产生DC，需要添加IL-4。用Flt3L和IL-4产生的DC在用肿瘤坏死因子-（TNF-）或CD40L刺激后发生成熟，其特征为CD83表达、MHC上调、黏附分子和共刺激分子增加以及同种异体增殖反应增强。相反，单独的CD40连接在缺乏GM-CSF和IL-4的情况下促进贴壁血单核细胞分化为功能性DC。这些细胞表现出成熟DC的所有表型和功能特征，并且在引发针对HIV肽的主要组织相容性复合体（MHC）I类限制性CTL反应中是有效的刺激细胞，而其呈递可溶性蛋白质抗原的能力降低。使用半定量RT-PCR，在所有产生的DC群体中均检测到DC-CK1和IL-18转录本，与所使用的生长因子无关。我们的研究结果为CD40-CD40L相互作用对于启动和维持T细胞反应的重要性提供了进一步证据，并证实了新出现的概念，即血单核细胞根据外部刺激提供DC的额外来源。
