Roles of free fatty acid macrophage mediated killing of mycobacteria

我们之前报道过活性氮中间体和游离脂肪酸（FFA）在小鼠腹膜巨噬细胞（Mφs）抗结核分枝杆菌（MTB）和鸟分枝杆菌复合体（MAC）的活性表达中起重要作用。本文研究了从膜磷脂中特异性释放花生四烯酸（AA）的磷脂酶A_2 （PLA_2）在m - φ抗细菌活性中的作用，得到了以下结果：(1)经IFN-γ活化的m - φs中，醌（PLA_2抑制剂）和a-TFMK （cPLA_2抑制剂）均能促进MTB的细胞内生长。(2)MTB感染m - φ后，在m - φ培养过程中，从负载^3H-AA的m - φ中回收的MTB生物的放射性逐渐增加。当m - φs被quinacrine或a- tfmk处理时，这种现象被阻断，这表明AA以cpla_2依赖的方式在吞噬体内转运到MTB。(3)在IFN-γ活化的Mφ中，cPLA_2和iNO5 mRNA的表达增加，而sPLA_2 mRNA的表达不增加。(4)用抗cPLA_2抗体和芘标记的cPLA_2底物对感染的m - φs进行荧光显微镜观察，发现cPLA_2在m - φs吞噬体内易位到分枝杆菌。(5)在小鼠腹膜Mφ中，MTB感染后Mφ培养初期，Mφ丝氨酸残基上出现了MAP激酶依赖性的cPLA_2磷酸化，至少在48 h内逐渐升高，而在MTB感染后较短时间内，RAW Mφ中出现了cPLA_2的组成性磷酸化。这些结果表明，cPLA_2在Mφs对MTB和MAC的抑菌活性表达中起重要作用。

We previously reported that reactive nitrogen intermediates and free fatty acid (FFA) play important roles in the expression of the activity of mouse peritoneal macrophages (Mφs) against Mycobacterium tuberculosis (MTB) and M. avium complex (MAC). In the present study, we examined the roles of phospholipase A_2 (PLA_2) which specifically releases arachidnic acid (AA) from membrane phospholipid in Mφantimycobacterial activity, and obtained the following results. (1) The intracellular growth of MTB residing in IFN-γ-activated Mφs was accelerated by the quinacrine (PLA_2 inhibitor) and a-TFMK (cPLA_2 inhibitor) (2) The radioactivity of MTB organisms, which were recovered from ^3H-AA loaded Mφs, was progressively increased during Mφ cultivation after MTB infection. Such a phenomenon was blocked when Mφs were treated quinacrine or a-TFMK, indicating that AA translocated to MTB within phagosome in a cPLA_2-dependent fashion. (3) The expression of cPLA_2 and iNO5 mRNAs was increased in IFN-γ- activated Mφ, this is not the case for sPLA_2 mRNA. (4) Fluorescence microscopy on infected Mφs stained with anti cPLA_2 antibody and pyrene labelled cPLA_2 substrates demonstrated that cPLA_2 translocated to mycobacterial organisms within the phagosomes of the Mφs. (5) In the case of mouse peritoneal Mφs, MAP kinase-dependent cPLA_2 phospholylation at the serine residues was observed in the early phase of Mφ cultivation after MTB infection, gradually increased at least for 48 h. On the other hand, constitutive phospholylation of cPLA_2 was observed in RAW Mφs regard less of MTB infection. These findings indicate that cPLA_2 plays important roles in the expression of antimicrobial activity of Mφs against MTB and MAC.