肺泡巨噬细胞在猪链球菌2型引致肺炎过程中的作用

The role played by alveolar macrophages in development of pneumonia caused by Streptococcus suistype 2

为探明肺巨噬细胞在猪链球菌2型(Streptococcus suis type 2,SS2)引致肺炎过程中发挥的作用,用SS2体外感染4周龄SPF巴马小型猪肺泡巨噬细胞(porcine alveolar macrophage,PAM),首先以10倍递增的SS2∶PAM感染比(MOI)分别为1∶10～1 000∶1感染12h;其次以MOI=100∶1分别感染1、2、4、6、12、24h,ELISA检测三种主要炎性细胞因子TNF-α、IL-1和IL-6的质量浓度及动态变化,同时光镜观察PAM的形态变化。结果显示,三种细胞因子具有相似的动态表达趋势,其表达量均随MOI升高和作用时间的延长而上升,与对照组之间均差异极显著(P<0.01),菌液浓度之间和时间点之间均差异显著(P<0.05);同时PAM的形态异常和崩解死亡比例依次增多,表明三种炎性因子的表达量与SS2的浓度和感染时间之间呈正相关;受损或濒死细胞比例亦正相关;提示三种炎性因子由受损细胞或濒死细胞产生。MOI为1∶1感染12h,或MOI为100∶1感染6h的条件下均可致70%的PAM崩解死亡,表明此条件下SS2可摧毁由PAM构筑的第一道防线。三种炎性细胞因子表达最高峰的条件是MOI为100∶1,感染12h,此时镜下细胞几乎全部崩解死亡,表明PAM的完全死亡伴随炎性因子的最大释放,炎症随吞噬功能的完全丧失而开始。结果表明,在SS2引致肺炎过程中,AM的作用是炎症的起始者,而不是抵御细胞。

In order to find out the role of alveolar macrophage（AMs） in development of pneumonia caused by Streptococcus suis type 2（SS2）,porcine alveolar macrophages（PAMs）,isolated from SPF Bamamini pigs of 4-week-old,were infected by SS2 in vitro.First,PAMs were infected by SS2 at the MOI（multiplicity of infection） from 1∶10 to 1 000∶1 for 12 h respectively.Secondly,PAMs were infected by SS2 at the MOI of 100∶1 for 1,2,4,6,12 and 24 h respectively.The dynamics of three major inflammatory cytokines,named as TNF-α,IL-1 and IL-6 produced by PAMs,were measured by ELISA kit.And the morphocytological changes of PAM were observed under light-microscopy at the same time.In result,the similar dynamics of the three major inflammatory cytokines.With the increase of MOI and infection time,both the levels of this three major inflammatory cytokines,TNF-α,IL-1 and IL-6,showed highly significant（P0.01） increase compared with negative control PAMs,and significant（P0.05） between every concentration and infection time,and the percentage of morphocytologically abnormal and necrotic PAMs increased as well indicating a positive correlation among MOI and infected duration,levels of TNF-α,IL-1 and IL-6,and rate of abnormal or necrotic PAMs,indicating that TNF-α,IL-1 and IL-6 were secreted by damaged even dying PAMs. Both conditions,MOI as 1∶1 for 12h,or MOI as 100∶1 for 6h,70% PAMs were broken,and the first defense line constituted by AMs were destroyed.With MOI increased up to 100∶1 for 12h,almost 100% PAMs lost structure,showing peaks of TNF-α,IL-1 and IL-6 and complete death of PAMs came together,implying the beginning of inflammation at the point of total abolishment of phagocytosis.The results showed that PAM was the initiator of pneumonia rather than defender in SS2 infection.