Cywes C, Godenir N L, Hoppe H C, Scholle R R, Steyn L M, Kirsch R E, Ehlers M R
Department of Medical Biochemistry, University of Cape Town Medical School, South Africa.
Infect Immun. 1996 Dec;64(12):5373-83. doi: 10.1128/iai.64.12.5373-5383.1996.
Nonopsonic invasion of mononuclear phagocytes by Mycobacterium tuberculosis is likely important in the establishment of a primary infection in the lung. M. tuberculosis binds to a variety of phagocyte receptors, of which the mannose receptor and complement receptor type 3 (CR3) may support nonopsonic binding. CR3, a beta2 integrin, is a target for diverse intracellular pathogens, but its role in nonopsonic binding remains uncertain. We have examined the binding of M. tuberculosis H37Rv to human CR3 heterologously expressed in Chinese hamster ovary (CHO) cells, thereby circumventing the problems of competing receptors and endogenously synthesized complement, which are inherent in studies with mononuclear phagocytes. The surface expression of CD11b and CD18 was assessed by immunofluorescence, immunobead binding, flow cytometry, and immunoprecipitation with anti-CD11b and anti-CD18 monoclonal antibodies (MAbs). The functional activity of the surface-expressed CD11b/CD18 (CR3) heterodimer was confirmed by rosetting with C3bi-coated microspheres. We found that M. tuberculosis bound four- to fivefold more avidly to CR3-expressing CHO cells than to wild-type cells and, importantly, that this binding was at similar levels in the presence of fresh or heat-inactivated human or bovine serum or no serum. In contrast, Mycobacterium smegmatis bound poorly to CR3-expressing CHO cells in the absence of serum, but after opsonization in serum, binding was comparable to that of M. tuberculosis. The binding of M. tuberculosis to the transfected CHO cells was CR3 specific, as it was inhibited by anti-CR3 MAbs, particularly the anti-CD11b MAbs LM2/1 (I domain epitope) and OKM1 (C-terminal epitope), neither of which inhibit C3bi binding. MAb 2LPM19c, which recognizes the C3bi-binding site on CD11b, had little or no effect on M. tuberculosis binding. The converse was found for the binding of opsonized M. smegmatis, which was strongly inhibited by 2LPM19c but unaffected by LM2/1 or OKM1. CR3-specific binding was also evidenced by the failure of M. tuberculosis to bind to CHO cells transfected with an irrelevant surface protein (angiotensin-converting enzyme) in the presence or absence of serum. We conclude that the binding of M. tuberculosis H37Rv to CR3 expressed in CHO cells is predominantly nonopsonic and that the organism likely expresses a ligand that binds directly to CR3.
结核分枝杆菌对单核吞噬细胞的非调理素依赖性侵袭在肺部原发性感染的建立过程中可能具有重要意义。结核分枝杆菌可与多种吞噬细胞受体结合，其中甘露糖受体和3型补体受体（CR3）可能支持非调理素依赖性结合。CR3是一种β2整合素，是多种细胞内病原体的作用靶点，但其在非调理素依赖性结合中的作用仍不明确。我们研究了结核分枝杆菌H37Rv与在中国仓鼠卵巢（CHO）细胞中异源表达的人CR3的结合情况，从而避免了单核吞噬细胞研究中固有的竞争性受体和内源性合成补体的问题。通过免疫荧光、免疫珠结合、流式细胞术以及用抗CD11b和抗CD18单克隆抗体（MAb）进行免疫沉淀来评估CD11b和CD18的表面表达。通过与C3bi包被的微球进行花环试验证实了表面表达的CD11b/CD18（CR3）异二聚体的功能活性。我们发现，结核分枝杆菌与表达CR3的CHO细胞的结合亲和力比与野生型细胞高4至5倍，重要的是，在存在新鲜或热灭活的人或牛血清或无血清的情况下，这种结合水平相似。相比之下，耻垢分枝杆菌在无血清时与表达CR3的CHO细胞结合较差，但在血清中调理后，其结合情况与结核分枝杆菌相当。结核分枝杆菌与转染的CHO细胞的结合具有CR3特异性，因为它受到抗CR3 MAb的抑制，特别是抗CD11b MAb LM2/1（I结构域表位）和OKM1（C末端表位），这两种抗体均不抑制C3bi结合。识别CD11b上C3bi结合位点的MAb 2LPM19c对结核分枝杆菌的结合几乎没有影响。对于调理后的耻垢分枝杆菌的结合则发现相反的情况，它受到2LPM19c的强烈抑制，但不受LM2/1或OKM1的影响。在有或无血清的情况下，结核分枝杆菌不能与转染了无关表面蛋白（血管紧张素转换酶）的CHO细胞结合，这也证明了CR3特异性结合。我们得出结论，结核分枝杆菌H37Rv与CHO细胞中表达的CR3的结合主要是非调理素依赖性的，并且该菌可能表达一种直接与CR3结合的配体。
