Nibbering P H, Broug-Holub E, Bezemer A C, Jansen R, van de Winkel J G, Geertsma M F
Dept. of Infectious Diseases, University Hospital, Leiden, The Netherlands.
Front Biosci. 1996 Apr 1;1:a25-33. doi: 10.2741/a102.
Phagocytes bear more than one class of receptors for the Fc domain of IgG (FcgammaR). In addition the same ligand can interact with different classes of FcgammaR. This complexity makes it difficult to study the contribution of the various classes of FcgammaR to antimicrobial functions. To circumvent this difficulty, in the present study mouse 3T6 fibroblasts transfected with cDNA encoding for human FcgammaR type IIa (FcgammaRIIa-expressing cells) were used to determine the role of this receptor in phagocytosis and intracellular killing of serum-opsonized Staphylococcus aureus. Experiments using microbiological and fluorescent techniques to discriminate between cell-adherent and intracellular bacteria revealed that serum-opsonized bacteria are phagocytized by FcgammaRIIa-expressing cells, but not by parental fibroblasts. Non-opsonized bacteria were poorly internalized by FcgammaRIIa-expressing as well as parental fibroblasts. Furthermore, incubation of FcgammaRIIa-expressing cells with opsonized bacteria at 4oC and incubation of FcgammaRIIa-expressing cells with cytochalasin E prior to addition of opsonized bacteria inhibited the phagocytosis of these bacteria almost completely. Phagocytosis of opsonized bacteria by FcgammaRIIa-expressing cells was partly inhibited by selective inhibition of protein tyrosine kinases (PTK). FcgammaRIIa cross-linking initiated transient tyrosine phosphorylation of various proteins in FcgammaRIIa-expressing cells. These data indicate that activation of PTK is involved in the FcgammaRIIa-mediated phagocytosis of opsonized S. aureus by transfected fibroblasts. Human serum from normal individuals and agammaglobulinemic patients triggered the intracellular killing of S. aureus by FcgammaRIIa-expressing fibroblasts. Surprisingly, heat-inactivated human serum, IgG and incubation with anti-FcgammaRII antibodies followed by a bridging secondary antibody did not stimulate the killing process. The possibility that these ligands did not interact with FcgammaRIIa on the cells can be excluded since they induced tyrosine phosphorylation of cellular proteins. The serum factor that stimulates the intracellular killing of bacteria by FcgammaRIIa-expressing cells is not yet identified. Oxygen-independent mechanisms are thought to be responsible for the killing of intracellular bacteria by these cells since the NADPH oxidase inhibitor diphenylene iodonium did not affect the serum-stimulated intracellular killing of S. aureus and no reactive oxygen and nitrogen intermediates were produced by FcgammaRIIa-expressing cells after appropiate stimulation. Taken together, these data show that phagocytosis but not intracellular killing of S. aureus is mediated via FcgammaRIIa on cells expressing this receptor.
吞噬细胞带有不止一类针对IgG Fc结构域的受体（FcγR）。此外，同一配体可与不同类别的FcγR相互作用。这种复杂性使得研究各类FcγR对抗菌功能的贡献变得困难。为了克服这一困难，在本研究中，使用转染了编码人FcγR IIa cDNA的小鼠3T6成纤维细胞（FcγRIIa表达细胞）来确定该受体在吞噬作用以及血清调理的金黄色葡萄球菌细胞内杀伤中的作用。使用微生物学和荧光技术区分细胞黏附菌和细胞内菌的实验表明，血清调理的细菌被FcγRIIa表达细胞吞噬，但未被亲代成纤维细胞吞噬。未调理的细菌被FcγRIIa表达细胞和亲代成纤维细胞摄取的效率都很低。此外，将FcγRIIa表达细胞与调理后的细菌在4℃孵育，以及在添加调理后的细菌之前将FcγRIIa表达细胞与细胞松弛素E孵育，几乎完全抑制了这些细菌的吞噬作用。FcγRIIa表达细胞对调理后细菌的吞噬作用部分受到蛋白酪氨酸激酶（PTK）选择性抑制的影响。FcγRIIa交联引发了FcγRIIa表达细胞中各种蛋白质的瞬时酪氨酸磷酸化。这些数据表明，PTK的激活参与了转染的成纤维细胞通过FcγRIIa介导的对调理后金黄色葡萄球菌的吞噬作用。来自正常个体和无丙种球蛋白血症患者的人血清引发了FcγRIIa表达的成纤维细胞对金黄色葡萄球菌的细胞内杀伤。令人惊讶的是，热灭活的人血清、IgG以及用抗FcγRII抗体孵育后再用桥连二抗孵育均未刺激杀伤过程。由于这些配体诱导了细胞蛋白的酪氨酸磷酸化，因此可以排除它们未与细胞上的FcγRIIa相互作用的可能性。刺激FcγRIIa表达细胞对细菌进行细胞内杀伤的血清因子尚未确定。由于NADPH氧化酶抑制剂二苯基碘鎓不影响血清刺激的对金黄色葡萄球菌的细胞内杀伤，且FcγRIIa表达细胞在适当刺激后未产生活性氧和氮中间体，因此认为细胞内细菌的杀伤是由不依赖氧的机制负责的。综上所述，这些数据表明，表达该受体的细胞上通过FcγRIIa介导的是金黄色葡萄球菌的吞噬作用而非细胞内杀伤。
