Mason K M, Bigley N J, Fink P S
Department of Microbiology and Immunology, Wright State University, Dayton, OH 45435, USA.
J Immunol Methods. 1998 Feb 1;211(1-2):147-58. doi: 10.1016/s0022-1759(97)00200-7.
We have developed a novel co-culture system in which murine splenocytes are cultured with live bacteria in the presence of a bacteriostatic antibiotic. Superantigens, like staphylococcal enterotoxin B (SEB) are important factors in bacterial pathogenicity. Research has shown that superantigens affect numerous immune cell types, either directly or indirectly, yet their involvement in pathogenic mechanisms remains poorly defined. In these studies, we utilize the co-culture system to study how superantigen pretreatment affects interferon-gamma (IFN-gamma) production by splenocytes co-cultured with gram-positive bacteria. Streptococcus mutans, S. sanguis and Bacillus subtilis were tested for susceptibility to a panel of antibiotics. Spectinomycin was found to maintain a bacteriostatic state of approximately 10(5) bacteria ml(-1) at optimal concentrations for each bacterial strain. Co-culturing splenocytes with bacteria did not affect splenocyte viability and cultured splenocytes responded to mitogenic stimulation as expected. Two days after SEB pretreatment, isolated splenocytes cultured with either Streptococcus species produced 10-15 times more IFN-gamma than splenocytes from sham-injected controls; however, no differences in CD4+ or CD8+ T cell populations appeared in cultures with or without bacteria. Splenocytes isolated four days after SEB treatment did not produce significant amounts of IFN-gamma in co-culture. Co-cultures containing live bacteria produced four times more IFN-gamma than cultures containing heat-killed bacteria. Splenocytes depleted of natural killer (NK) cells prior to SEB treatment produced 25% less IFN-gamma after 20 h co-culturing with S. mutans. T lymphocytes were identified to be the major producer of IFN-gamma at this time point by intracellular cytokine staining. Apparently SEB exposure primes a response to live bacteria and the response is evident two days after initial exposure. The in vitro co-culture system allows us to observe host responses to bacteria in the context of the multicellular interdependent immune response. With this assay we can more closely 'mimic' in vivo events, particularly immune cell interactions in microfloral environments, to study how the pathogenic effects of superantigens alter this response.
我们开发了一种新型共培养系统，其中小鼠脾细胞在抑菌抗生素存在的情况下与活细菌一起培养。超抗原，如葡萄球菌肠毒素B（SEB），是细菌致病性的重要因素。研究表明，超抗原直接或间接影响多种免疫细胞类型，但其在致病机制中的作用仍不清楚。在这些研究中，我们利用共培养系统研究超抗原预处理如何影响与革兰氏阳性菌共培养的脾细胞产生干扰素-γ（IFN-γ）。测试了变形链球菌、血链球菌和枯草芽孢杆菌对一组抗生素的敏感性。发现壮观霉素在每种细菌菌株的最佳浓度下可维持约10(5) 个细菌毫升(-1) 的抑菌状态。脾细胞与细菌共培养不影响脾细胞活力，培养的脾细胞对有丝分裂原刺激的反应符合预期。SEB预处理两天后，与任何一种链球菌共培养的分离脾细胞产生的IFN-γ 比假注射对照的脾细胞多10至15倍；然而，在有或没有细菌的培养物中，CD4+ 或CD8+ T细胞群体没有差异。SEB处理四天后分离的脾细胞在共培养中未产生大量IFN-γ。含有活细菌的共培养物产生的IFN-γ 比含有热灭活细菌的培养物多四倍。在SEB处理前耗尽自然杀伤（NK）细胞的脾细胞在与变形链球菌共培养20小时后产生的IFN-γ 减少25%。通过细胞内细胞因子染色确定此时T淋巴细胞是IFN-γ 的主要产生者。显然，SEB暴露引发了对活细菌的反应，并且在初次暴露两天后反应明显。体外共培养系统使我们能够在多细胞相互依赖的免疫反应背景下观察宿主对细菌的反应。通过这种测定，我们可以更密切地“模拟”体内事件，特别是微生物环境中的免疫细胞相互作用，以研究超抗原的致病作用如何改变这种反应。
