Toll-like receptors是近年來被發現在辨識微生物並活化免疫及發炎反應上重要的感應器。未甲基化（unmethylated）胞嘧啶（cytosine）-鳥糞鹼（guanine）雙核甘酸序列（CpG motifs），有強力免疫刺激的能力，在細菌中遠大於脊椎動物。而未甲基化CpG序列能藉由Toll-like receptor-9 (TLR9)去啟動先天性免疫並活化一連串訊息傳遞，引發許多生物體的反應，包含巨噬細胞移行。在我們的研究中，證明了在CpG刺激下，Raw264.7巨噬細胞的移行和總體酪氨酸磷酸化明顯增加。但合併處理PP2（Src family kinases [SFKs] 的抑制劑）後，細胞的移行減少，透露出SFKs參與在CpG誘發的細胞移行中。進一步發現，Raw264.7細胞在CpG刺激下，只有Src表現增加，Lyn、Fgr和Hck蛋白量沒有很大的差異，而取自老鼠腹腔之巨噬細胞也有相同的現象。在Raw264.7細胞用RNA interference的技術減少Src表現量，會減弱CpG誘發的細胞移行，而回復Src表現，則可恢復細胞移行能力。合併處理TLR9抑制劑chloroquine和quinacrine後，CpG誘發的Src表現和細胞移行皆減弱，也顯示此過程為TLR9-dependent。另外，我們也發現在缺少iNOS的巨噬細胞，其CpG引發的Src表現和細胞移行皆減少。由上述的結果，我們認為iNOS可以調節Src表現，進而影響CpG誘發的細胞移行能力。
關鍵字：CpG 雙核甘酸序列，Src，巨噬細胞移行能力
Toll-like receptors (TLRs) have recently been identified as important sensors of infection that, upon microbial recognition, activate the immune and inflammatory responses. DNA containing cytosine-guanine dinucleotide (CpG) motifs have potent immunostimulatory activities. Unmethylated CpG dinucleotides in particular sequence contexts occur frequently in bacterial but not in vertebrate DNA. Unmethylated CpG motifs initiate innate immune responses via Toll-like receptor-9 (TLR9), which activates a spectrum of signaling molecules and triggers a variety of biological responses including migration in macrophages. In this study, we demonstrated that upon CpG stimulation, the motility and the content of total protein tyrosyl phosphorylation in Raw264.7 macrophages were greatly enhanced. The abrogation of CpG-induced migration by PP2 (an inhibitor for Src family kinases [SFKs]) suggested the involvement of SFKs in this process. Analysis of the expression of SFKs in Raw264.7 cells revealed that only the expression of Src (but not Lyn, Hck and Fgr) was CpG-inducible. And similar result was also detected in rat peritoneal macrophages. Remarkably, silencing of Src expression by RNA interference reduced CpG-evoked cell mobilization in Raw264.7 macrophages and restored Src expression could rescue this defect. The inhibition of CpG-induced Src expression and migration in cells pretreated with chloroquine and quinacrine implicating these events were TLR9-dependent. Notably, this CpG-mediated mobilization and Src induction was suppressed in macrophages devoid of iNOS. With these findings, we concluded that iNOS-induced Src expression played a significant role in CpG-elicited macrophage migration.
Key word：CpG motifs, Src, macrophage migration
