Department of Immunology and Molecular Immunology and BK21 CLS Program, School of Dentistry, Seoul National University, Seoul 110-749, Republic of Korea.
Mol Immunol. 2011 Sep;48(15-16):1966-74. doi: 10.1016/j.molimm.2011.05.026. Epub 2011 Jun 22.
The molecular mechanisms underlying the non-opsonic phagocytosis of bacteria by neutrophils are poorly understood. We previously reported the efficient uptake of Streptococcus sanguinis by human neutrophils in the absence of opsonins. To characterize the phagocytosis receptor, protein lysates from neutrophils and HL-60 cells were subjected to affinity chromatography using epoxy beads coated with S. sanguinis. Denaturing electrophoresis of the eluted proteins and subsequent mass spectrometry revealed that one of the proteins eluted from neutrophils was proteinase 3 (PR3). Enzymatic cleavage of the glycosylphosphatidylinositol linker of NB1, a co-receptor for membrane-bound PR3 (mPR3), significantly reduced the phagocytosis of S. sanguinis. In addition, the neutralization of mPR3 with antibody reduced both binding and phagocytosis of S. sanguinis. Treatment of neutrophils with a serine proteinase inhibitor indicated that protease activity is required for phagocytosis. Thus, we studied whether protease-activated receptor 2 (PAR2) is involved in signal transmission from mPR3 during this process. Indeed, neutralizing antibodies against PAR2 inhibited phagocytosis and S. sanguinis-induced calcium mobilization desensitized PAR2. Furthermore, the phagocytosis of S. sanguinis and the concomitant activation of Rho family GTPases were inhibited by the intracellular calcium chelator, BAPTA-AM. Collectively, mPR3 acts as a non-opsonic phagocytosis receptor for bacteria probably by activating PAR2 in neutrophils.
中性粒细胞非调理吞噬细菌的分子机制尚不清楚。我们之前曾报道过无调理素存在的情况下人中性粒细胞对酿脓链球菌的有效摄取。为了鉴定吞噬受体，我们用酿脓链球菌包被的环氧珠对中性粒细胞和 HL-60 细胞的蛋白裂解物进行亲和层析。洗脱蛋白的变性电泳和随后的质谱分析显示，从中性粒细胞洗脱的一种蛋白是蛋白酶 3（PR3）。NB1 糖基磷脂酰肌醇连接物的酶切，NB1 是膜结合 PR3（mPR3）的共受体，显著降低了酿脓链球菌的吞噬作用。此外，用抗体中和 mPR3 减少了酿脓链球菌的结合和吞噬作用。用丝氨酸蛋白酶抑制剂处理中性粒细胞表明蛋白酶活性是吞噬作用所必需的。因此，我们研究了在这个过程中蛋白酶激活受体 2（PAR2）是否参与 mPR3 的信号转导。事实上，针对 PAR2 的中和抗体抑制了吞噬作用和酿脓链球菌诱导的钙动员使 PAR2 脱敏。此外，酿脓链球菌的吞噬作用和 Rho 家族 GTP 酶的伴随激活被细胞内钙螯合剂 BAPTA-AM 抑制。总之，mPR3 可能通过激活中性粒细胞中的 PAR2 作为细菌的非调理吞噬受体发挥作用。
