Raman Thiagarajan, Arumugam Munusamy, Mullainadhan Periasamy
Laboratory of Pathobiology, Department of Zoology, University of Madras, Guindy Campus, Chennai 600 025, India.
Fish Shellfish Immunol. 2008 Mar;24(3):337-45. doi: 10.1016/j.fsi.2007.12.005. Epub 2008 Jan 28.
Hemocyte mediated phagocytosis is one of the vital components of innate defence mechanisms in crustaceans and this phagocytic process is aided by serum agglutinins. However, literature on agglutinin mediated opsono-phagocytosis is unclear in the case of Macrobrachium rosenbergii hemocytes. Further, very few studies in the case of superoxide anion generation and none with regard to nitric oxide generation during phagocytosis exist among crustaceans. We investigated the occurrence of agglutinins in the serum and the role of serum agglutinins in mediating phagocytosis by the hemocytes. We show that the prawn serum possesses agglutinins that function as opsonins during phagocytosis of HB RBC by the hemocytes. Hemagglutination-inhibition assays revealed the specificity of serum agglutinins for N-acetylated hexoses, namely GalNAc, GlcNAc and ManNAc, with a higher affinity for ManNAc. In addition, ManNAc was able to inhibit the phagocytic response (by about 60%) of the hemocytes against serum pretreated HB RBC, wherein the serum was previously treated with ManNAc. We next investigated the ability of the hemocytes to generate superoxide anion and nitric oxide during HB RBC phagocytosis and results show generation of both these free radicals. In addition, there was an enhancement in generation (75% increase) of these free radicals during agglutinin mediated opsonophagocytosis, when compared to buffer treated targets and interestingly this enhanced generation was inhibited by ManNAc (27% for superoxide anion and 36% for nitric oxide), an inhibitory sugar for phagocytosis. Inhibition of phagocytosis induced superoxide anion generation by DPI (53%), sodium azide (56%) and tropolone (61%), reveals the possible involvement of NADPH-oxidases, peroxidases and probably phenoloxidases, respectively, in the generation of superoxide anion. Similarly, decrease in nitric oxide generation in the presence of l-NIO (47%) during phagocytosis lends support to the role of nitric oxide generation during cellular immune processes. These findings thus suggest a role for superoxide anion and nitric oxide in the innate defense mechanism, namely phagocytosis, in Macrobrachium rosenbergii.
血细胞介导的吞噬作用是甲壳类动物先天防御机制的重要组成部分之一，这一吞噬过程由血清凝集素辅助。然而，关于罗氏沼虾血细胞中凝集素介导的调理吞噬作用的文献尚不清楚。此外，在甲壳类动物中，关于吞噬过程中超氧阴离子生成的研究很少，关于一氧化氮生成的研究则尚无报道。我们研究了血清中凝集素的存在情况以及血清凝集素在介导血细胞吞噬作用中的作用。我们发现，对虾血清中含有凝集素，这些凝集素在血细胞吞噬HB红细胞的过程中起调理素的作用。血凝抑制试验揭示了血清凝集素对N-乙酰化己糖（即GalNAc、GlcNAc和ManNAc）的特异性，其中对ManNAc的亲和力更高。此外，ManNAc能够抑制血细胞对经ManNAc预处理血清的HB红细胞的吞噬反应（约60%）。接下来，我们研究了血细胞在吞噬HB红细胞过程中产生超氧阴离子和一氧化氮的能力，结果显示这两种自由基均有产生。此外，与缓冲液处理的靶标相比，在凝集素介导的调理吞噬作用过程中，这些自由基的产生有所增强（增加75%），有趣的是，这种增强的产生被ManNAc抑制（超氧阴离子为27%，一氧化氮为36%），ManNAc是吞噬作用的抑制性糖。DPI（53%）、叠氮化钠（56%）和托酚酮（61%）对吞噬作用诱导的超氧阴离子生成的抑制作用，分别揭示了NADPH氧化酶、过氧化物酶以及可能的酚氧化酶可能参与了超氧阴离子的生成。同样，在吞噬过程中，l-NIO存在时一氧化氮生成的减少（47%）支持了一氧化氮在细胞免疫过程中的作用。因此，这些发现表明超氧阴离子和一氧化氮在罗氏沼虾的先天防御机制即吞噬作用中发挥作用。
