中性粒细胞诱导适应性免疫应答过程中树突状细胞成熟活化

目的 探讨T辅助（Th）1细胞免疫应答过程中,中性粒细胞（PMN）对树突状细胞（DC）的调节作用。方法 将脂多糖（LPS）刺激的中性粒细胞（LPS-PMN）和未成熟树突状细胞（imDC）共培养,流式细胞术检测DC表面CD40和CD86,酶联免疫吸附试验（ELISA）检测白细胞介素12（IL-12）、肿瘤坏死因子α（TNF-α）。将与LPS—PMN共培养后DC（PMN-DC）提纯,与FITC标记的卵清蛋白（FITC-OVA）培养,检测其吞噬功能。将PMN-DC与D011．10T细胞和OVA17肽共培养,计活细胞数,胞内染色测干扰素γ（IFN-γ）、白细胞介素4（IL-4）。结果 LPS-PMN可刺激imDC CD40和CD86上调并分泌IL-12、肿瘤坏死因子（TNF）-α,且这种能力能被抗TNF-α单抗所抑制。与imDC对照,PMN—DC吞噬功能下降,能显著刺激D011．10T细胞增殖,分泌高水平IFN-γ、少量IL-4。结论 LPS-PMN具有促使imDC成熟活化,刺激其分泌细胞因子,发挥其在Th1免疫应答中抗原呈递,促进Th1分化的作用。

Neutrophils induce the maturation and activation of immature dendritic cells during adaptive immune responses

OBJECTIVE: To investigate whether polymorphonuclear neutrophilic leucocyte (PMN) can induce the maturation and activation of immature dendritic cells, thus promoting the Th1 immunity to microbial pathogens in vitro. METHODS: PMN isolated from mouse bone marrow were stimulated by lipopolysaccharide (LPS), and were cultured with immature DC (imDCs) isolated from mouse bone marrow for 18 hours. Then the analysis of DC surface markers CD40 and CD86 was done by flow cytometry, and ELISA was used to detect the levels of TNF-alpha and IL-12. Purified DC cocultured with LPS-PMN (PMN-DC) and imDC were incubated with fluorescein isothiocyanate conjugated OVA (FITC-OVA), and the phagocytic capacity of PMN-DC and imDC was assessed by flow cytometry. Splenic CD4(+) T cells from DO11.10 x C57BL/6 F1 hybrid mice were obtained, and then cultured with PMN-DC or imDC in the presence of OVA (323 - 339). The cells were double stained with anti-CD4-PE and 7-amino-actinomycin D. The cellular data were acquired for 56s with a flow cytometer and the assay of IFN-gamma and IL-4 was done by intracellular staining. RESULTS: LPS-PMN induced strong up-regulation of CD40 and CD86, and this capacity was remarkably inhibited after being neutralized by anti-TNF-alpha monoclonal antibody. LPS-PMN also stimulated imDCs to produce IL-12 and TNF-alpha. PMN-DCs demonstrated decreased phagocytic capacity compared with that of imDCs. Furthermore, the PMN-DC induced considerable DO11.10 T cell proliferation, and stimulated DO11.10 T cell to produce a large amount of IFN-gamma, but a relatively low amount of IL-4. CONCLUSION: LPS-PMN can induce the maturation and activation of imDCs, including the cytokine secretion, specific antigen presentation and Th1 differentiation, thus promoting the type I immunity to microbial pathogens.