增加衰变加速因子可改善实验性自身免疫性脑脊髓膜炎

衰变加速因子（Decay accelerating factor，DAF）作为一种位于细胞膜表面的补体调节成分，不仅可阻止C3和C5转化酶的装配，并且可通过诱导催化亚单位C2a的快速解离而使已形成的C3、C5转化酶失去稳定性，进而抑制C3a和C5a（前炎性成分）的产生。     

补体激活调节因子作为补体抑制剂的研究进展

补体激活调节因子 (RCA)家族的补体调节蛋白可阻断人类疾病时补体的不适当激活。它们均作用于补体前端反应 ,通过其短同源重复 (SCR)与C3b或C4b结合 ,或促进C3和C5转化酶的衰变 ,或辅助Ⅰ因子裂解C3b和C4b ,从而达到抑制补体激活的目的。本文对近年来RCA家族中部分成员作为补体抑制剂的研究进展作一综述。     

人红细胞膜衰变加速因子的分离纯化及其生物学活性鉴定

目的　获得纯化并具有生物学活性的人红细胞膜衰变加速因子。方法　经胰蛋白酶消化、正丁醇抽提及DE32和Sepharose 6B色谱分离等步骤从人红细胞膜影中分离纯化人红细胞膜衰变加速因子 (DAF)。以SDS PAGE及免疫印迹实验检测纯度及抗原特异性 ,以C3转化酶体外组装实验及促衰变活性实验检测其补体抑制活性。结果　 4 0 0mL人全血最终可得纯化DAF约 370 μg ,回收率达 10 .4 % ;比活性为每毫克 2 .2 4× 10 5units;纯化产物在SDS PAGE表现为 70 0 0 0u的单一蛋白条带 ,在免疫印迹实验中可与抗人DAF单抗特异性结合。在生物学活性实验中 ,纯化产物既可促进C3转化酶衰变 ,也可抑制C3转化酶形成。结论　采用本方法可获得纯化的具有生物学活性的人衰变加速因子     

中国人HLA—Ⅲ（C4A,C4B,Bf,C2）的遗传多态性及其与疾病的关系

C4、Bf、C2均为补体的固有成份,为形成两条途径C3转化酶与C5转化酶的关键分子。这三个补体蛋白的基因紧密连锁,定位于人第6染色体短臂(6p21.3),嵌在人MHC     

B因子——补体第二激活途径中的一个关键分子

补体是由20余种蛋白质构成的一个复杂的限制性蛋白解(limited proteoIysis)系统,藉蛋白解作用可将没有活性的天然补体成份裂解激活,级联式地进行下去,发挥抗感染,免疫调控等作用,在某些情况下也可造成组织损伤等病理反应。补体的激活途径有两条,分别称为第一(经典)途径与第二(旁路)途径。在这两条激活途径中,有两个结构与功能类似的丝氨酸蛋白酶原,分别参与第一及第二途径途径活化的关键步骤——C3转化酶与C5转化酶的形成,前者为C2,后者即B因子。本文就后者的目前研究进展结合我们在这方面做的一点工作作一简略回顾。     

补体激活调节因子作为补体抑制剂的研究进展

补体激活调节因子（ＲＣＡ）家族的补体调节蛋白可阻断人类疾病时补体的不适当激活。它们均作用于补体前端反应,通过其短同源重复（ＳＣＲ）与Ｃ３ｂ或Ｃ４ｂ结合,或促进Ｃ３和Ｃ５转化酶的衰变,或辅助Ⅰ因子裂解Ｃ３ｂ和Ｃ４ｂ,从而达到抑制补体激活的目的。本文对近年来ＲＣＡ家族中部分成员作为补体抑制剂的研究进展作一综述。     

补体激活第三途径——凝集素途径

MBL与C1q、MASP-1和MASP-2与C1r和C1s具有同源的结构和功能。MBL与MASP-1、MASP-2组成类似C1的复合物。MBL识别相应糖结构后,MASP-1和MASP-2相继激活,裂解C4和C2生成C3转化酶C4b2b,活化MASP-1还直接裂解C3,由此形成补体激活第三途径即凝集素途径。作为第一线天然免疫防御系统,该途径在种系进化、个体发育及病原体感染的早期均具十分重要的生物学意义。     

抗凝剂、温度和性别对体外中性粒细胞自发性激活的影响

从细胞形态学和细胞酶组织化学的角度出发，用伪足法和ＮＢＴ法检测体外中性粒细胞自发性激活，研究了抗凝剂、孵育温度和性别对检测结果的影响。发现：（１）ＥＤＴＡ可显著抑制中性粒细胞自发性激活，血标本以小剂量肝素抗凝为宜：（２）孵育温度对中性粒细胞自发性激活有影响，３７℃比２５℃的激活百分率高，同激活百分率出现较２５℃早１ｈ；（３）女性中性粒细胞自发性激活百分率较男性高，同激活百分率出现较男性早１ｈ。研究     

血清补体含量降低的临床意义

已知补体系统由20余种血浆蛋白构成,约占血清球蛋白总量的10%。经典途径包括五种蛋白:Clq、Clr、Cls、C4和C2。激活剂如IgG、IgM类免疫复合物与Clq结合,起动CI激活,然后激活C4、C2,形成经典途径C3     

C2——补体第一激活途径中的一个重要分子

一、C2的功能补体两条途径激活的关键步骤都是C3转化酶的形成。C2是补体第一前端反应中C3转化酶的酶原部分。当C1激活后形成丝氨酸蛋白酶Cī,它可将C4裂解为C4b 及C4a 大、小两个分段。C4b 上具有C2的结合位,两者结合成C4bC2。其中C2又可受到Cī的一次裂解,分出C2b 入液相,余下的C4b2a 即第一途径C3转化酶。在细胞表面C3b 存在的情况下,C4b2a 即呈C4b2a3b 的形式存在,此即C5转化酶。C2在血中的浓度极低,仅20mg/L,比B 因子低15倍,比C4低20倍,加之C4b2a 复合物上C2a 解离迅速,因此它在补体第一激活途径中是最早的限速步骤。     

Mechanism of activation of the classical pathway of complement by monoclonal IgE (DES). Restricted regulation of C4b by C4b-binding protein

A human monoclonal IgE from patient DES, IgE (DES), has been shown to activate the classical pathway of complement. The mechanism of this activation has been investigated and can be summarized as follows: (a) IgE (DES) is able to bind and activate C1 in a dose-dependent fashion. This activation increases with the size of the aggregates used, but the affinity of C1 for IgE (DES) is weaker than for IgG. (b) A classical pathway C3 convertase can be assembled on IgE (DES) using purified C1, C4 and C2. The formation decay of this convertase is similar to that formed on IgG with an half-life of 9 min at 37 degrees C. (c) The extrinsic regulation of the C3 convertase by C4bp is restricted on IgE (DES) as compared to IgG. This restriction is shown on both the formation and the decay of the convertase. The mechanism of activation of the classical pathway of complement by IgE (DES) thus present some similarities with the assembly of the C3 convertase by the alternative pathway.     

Inhibition of the alternative pathway of complement by glomerular chondroitin sulphate proteoglycan.

Cultured rat glomerular epithelial cells (GEC) synthesize and secrete a complement inhibitory chondroitin sulphate B proteoglycan (termed GCRF). As proteoglycans and their component glycosaminoglycans may affect several different steps of complement activation, the functional properties of GCRF were investigated in this study. GCRF inhibited preformed alternative pathway convertases (C3bBbP), but did not substantially accelerate their decay. In contrast to other polyanions, GCRF did not affect the activity of human or rat factor H, nor did it inhibit the terminal complement proteins. GCRF inhibited the effect of decay-accelerating factor (DAF) on C3bBbP when the two were incubated together and DAF was in excess, but it did not affect the decay of DAF of classical pathway convertases. However, when DAF was first incorporated into the erythrocyte membrane, the effect of GCRF on C3bBbP was additive to that of DAF. Thus, GCRF inhibits the activity of C3bBbP and blocks the action of DAF, but not that of factor H, perhaps by binding to factor B in the alternative pathway convertase.     

gp 58/68, a parasite component that contributes to the escape of the trypomastigote form of T. cruzi from damage by the human alternative complement pathway

A glycoprotein of apparent molecular weight 58,000 (unreduced)/68,000 (in its reduced form) (gp 58/68), which is one of the fibronectin/collagen receptors of Trypanosoma cruzi, was purified to homogeneity from the trypomastigote forms of the Tehuantepec and Y strains of the parasite. Purified gp 58/68 inhibited formation of cell-bound and fluid-phase alternative pathway C3 convertase in a dose-dependent fashion, as assessed using purified human complement components. Gp 58/68 differed from the human regulatory proteins H, DAF, MCP and CR1 and from previously reported regulatory proteins on the parasite membrane in that it was unable to enhance decay-dissociation of preformed alternative pathway C3 convertase sites, did not serve as a co-factor for I-mediated cleavage of C3b and had no inhibitory activity on the classical pathway convertases. The inhibitory effect of gp 58/68 was most likely dependent on an interaction of the protein with factor B rather than with C3b. Gp 58/68 provides trypomastigotes with an additional potential mechanism for escaping complement lysis by the human alternative pathway.     

Protection of the classical and alternative complement pathway C3 convertases, stabilized by nephritic factors, from decay by the human C3b receptor

Formation and function of the classical (C4b,2a) and alternative (C3b,Bb) complement pathway C3 convertases are regulated by the intrinsic lability of the enzymes, extrinsic decay by C4bp and H, cleavage of C4b and C3b by I, and by the inhibitory action of the C3b receptor molecule (CR1). Binding of C4 nephritic factor (C4Nef) to C4b and of C3 nephritic factor (C3Nef) to C3b stabilizes the C3 convertases and bypasses inactivation by C4bp, H and/or I. In the present study, binding of C4Nef to the classical C3 convertase was found to prevent decay of C4b,2a by inputs of CR1 that were at least 15 times the amount of CR1 which inactivated 50% unstabilized classical pathway C3 convertase sites in 2.5 min. CR1 could however inhibit lysis of C4b,2a(C4Nef)-bearing cells in a dose-dependent manner. The latter inhibitory effect was directed at the interaction of C5 with the C5 convertase, most likely at C5 binding to cell-bound C3b. In an analogous manner to C4Nef in the classical pathway, stabilization of alternative pathway C3b,Bb convertase sites by C3Nef resulted in a relative protection of C3 convertase sites from decay by CR1. Thus, C4Nef and C3Nef can bypass all mechanisms susceptible to regulate function of the classical and alternative pathway C3 convertases. Because CR1 is essential for degradation of C3b bound to immune complexes in whole blood, stabilization of C4b,2a and C3b,Bb by C4Nef and C3Nef may alter in vivo processing of immune complexes in patients with nephritic factors.     

Stringent regulation of complement lectin pathway C3/C5 convertase by C4b-binding protein (C4BP)

The complement lectin pathway, an essential component of the innate immune system, is geared for rapid recognition of infections as each C4b deposited via this pathway is capable of forming a C3/C5 convertase. In the present study, role of C4b-binding protein (C4BP) in regulating the lectin pathway C3/C5 convertase assembled on zymosan and sheep erythrocytes coated with mannan (E_Man) was examined. While the C4BP concentration for inhibiting 50% (IC₅₀) formation of surface-bound C3 convertase on the two surfaces was similar to that obtained for the soluble C3 convertase (1.05 nM), 3- and 41-fold more was required to inhibit assembly of the C5 convertase on zymosan (2.81 nM) and E_Man (42.66 nM). No difference in binding interactions between C4BP and surface-bound C4b alone or in complex with C3b was observed. Increasing the C4b density on zymosan (14,000–431,000 C4b/Zym) increased the number of C4b bound per C4BP from 2.87 to 8.23 indicating that at high C4b density all seven α-chains of C4BP are engaged in C4b-binding. In contrast, the number of C4b bound per C4BP remained constant (3.79 ± 0.60) when the C4b density on E_Man was increased. The data also show that C4BP regulates assembly and decay of the lectin pathway C3/C5 convertase more stringently than the classical pathway C3/C5 convertase because of a 7- to 13-fold greater affinity for C4b deposited via the lectin pathway than the classical pathway. C4BP thus regulates efficiently the four times greater potential of the lectin pathway than the classical pathway in generating the C3/C5 convertase and hence production of pro-inflammatory products, which are required to fight infections but occasionally cause pathological inflammatory reactions.     

Epstein-Barr virus transformed B cell lines derived from patients with systemic lupus erythematosus produce a nephritic factor of the classical complement pathway

Nephritic factor of the classical complement pathway (C4NeF) is an IgG antibody which stabilizes the C3 convertase (C4b2a) and has been detected in sera from patients with systemic lupus erythematosus (SLE) and acute postinfectious glomerulonephritis. In order to study the production of nephritic factor (NeF), mononuclear cells were isolated from the peripheral blood of patients with SLE and infected with Epstein-Barr virus (EBV) to establish active B lymphocyte cell lines. Supernatants from 15 established B cell lines, as well as from 10 B cell lines established from normal individuals, were investigated for their ability to conserve the classical and the alternative pathway C3 convertases as assessed by EAC3bBb and EAC14b2a stabilizing assays. Supernatants from 2 of 15 B cell lines from patients with SLE, but none from normal individuals, stabilized the classical C3 convertase without having any effect on the alternative pathway C3 convertase. Using anti-human Ig affinity chromatography, we showed that C4NeF activity resided in the IgG fraction; the IgG fraction containing C4NeF activity bound to the C4b2a complex, but not to C4b alone. On gel electrophoresis, following reduction, the heavy chains were slightly heavier than the heavy chains of normal IgG. We were able to isolate C4NeF from the sera of the 2 patients with SLE from whom the positive supernatants were derived, but were unable to detect any C4NeF activity in the sera of the other 13 patients and the 10 normal individuals. Serum and B cell line supernatant-derived C4NeF exhibited comparable characteristics. We conclude that C4NeF produced in vitro by EBV-transformed B cell lines derived from patients with SLE is functionally similar to the conventional C4NeF in serum. These studies confirm the production of autoantibodies by B cells with the ability to stabilize the classical pathway C3 convertase in certain patients with SLE; stabilization of the C4b2a enzyme in these patients is an apparent mechanism for the development of hypocomplementemia. Finally, preparation of homogeneous C4NeF in vitro should improve our understanding of the role of autoantibodies in complement metabolic disturbances in autoimmune diseases.     

New perspectives on mannan-binding lectin-mediated complement activation

The complement system is an important part of the innate immune system, mediating several major effector functions and modulating adaptive immune responses. Three complement activation pathways exist: the classical pathway (CP), the alternative pathway (AP), and the lectin pathway (LP). The LP is the most recently discovered, and least characterized. The CP and the LP are generally viewed as working through the generation of the C3 convertase, C4bC2b, and are here referred to as the "standard" pathways. In addition to the standard CP and LP, so-called bypass pathways have also been reported, allowing C3 activation in the absence of components otherwise believed critical. The classical bypass pathways are dependent on C1 and components of the AP. A recent study has shown the existence also of a lectin bypass pathway dependent on mannan-binding lectin (MBL) and AP components. The emerging picture of the complement system is more that of a small "scale-free" network where C3 acts as the main hub, than that of three linear pathways converging in a common terminal pathway.     

Mouse Crry/p65 is a regulator of the alternative pathway of complement activation

Like man, mouse has evolved a unique set of regulatory proteins which provide protection from complement-mediated damage to self membranes. The recently described mouse protein Crry/p65 has been shown to inhibit classical complement pathway C3 deposition on cell membranes in which it is expressed. In two distinct experimental systems, we now further delineate the regulatory activity of Crry/p65 and demonstrate its inhibitory effect on alternative complement pathway C3 activation. First, significant inhibition of mouse alternative pathway C3 deposition was demonstrated on neuraminidase-treated human K562 cells expressing recombinant Crry/p65. Second, using a baculovirus technique, recombinant Crry/p65 was synthesized as a soluble molecule and then purified. This molecule was found to inhibit mouse C3 deposition on the surface of zymosan, a potent alternative complement pathway activator. These studies, combined with our earlier findings, demonstrate that Crry/p65 can regulate both the classical and alternative complement pathways. Crry/p65 must, therefore, exert its effects prior to, or at the level of, the C3 convertases, in a fashion similar to that of human membrane cofactor protein and/or decay-accelerating factor. These studies provide further proof of the hypothesis that Crry/p65 is an evolutionarily unique, complement regulatory protein which has developed in mouse.     

Complement modulatory activity of bisbenzylisoquinoline alkaloids isolated from Isopyrum thalictroides--I. Influence on classical pathway in human serum.

Eleven bisbenzylisoquinoline alkaloids (BBI) were isolated from the plant Isopyrum thalictroides (L.). Treatment of normal human serum (NHS) with BBI resulted in a diminution of the haemolytic activity of the classical pathway (CP). The mode of action of the main alkaloids isopyruthaline (It1), fangchinoline (It2) and isotalictrine (It3) on CP activation was investigated in vitro. The inhibition was time- and temperature-related and for Itl and It3 depended on the concentration of Ca2+ and Mg2+ ions. It was established that the substances reduced C1 haemolytic activity. It2 and It3 enhanced the complement consumption caused by heat aggregated human IgG (HAGG). The BBI prevented the formation of C3 convertase of the classical pathway. The loss of haemolytic activity was partially restored by the addition of C142 reagent (zymosan-treated guinea pig serum) to alkaloids-treated NHS. The addition of the late components C3-9 (EDTA-treated rat sera) recovered to some extent the haemolytic activity of It1-treated NHS, but not of It2- and It3-treated NHS.     

Role of the C3b-binding site on C4b-binding protein in regulating classical pathway C5 convertase

A high affinity C5 convertase is generated when a C3 convertase deposits additional C3b molecules on and around itself thereby switching the substrate specificity of C3 convertase from C3 to C5. In the present study the role of the additional C3b molecules in influencing the regulation of classical pathway C5 convertase by C4b-binding protein (C4BP) was examined and compared to its precursor, the C3 convertase. Determination of IC(50) for inhibiting formation of the high affinity C5 convertase and for enhancing its decay (72 and 20 nM) were found to be similar to those obtained for the surface-bound C3 convertase (35 and 11 nM). No difference was observed in the cofactor activity of C4BP for surface-bound C4b alone or when in complex with C3b. Analysis of binding interactions between C4BP and EAC1,C4b cells revealed an average apparent dissociation constant (12 nM) similar to that obtained with EAC1,C4b cells with C3b on them (11 nM). Increasing the C4b or C3b density on the cell surface did not alter the affinity of C4BP. The data suggest that C4BP regulates the C5 convertase by mechanisms similar to those observed for the C3 convertase. Since the IC(50) for inhibiting formation of the soluble C3 convertase (5 nM) is 50-80-fold below the normal serum concentration of C4BP (250-400 nM), C4BP in blood effectively prevents formation of classical pathway C3 convertase in the fluid phase. Although deposition of additional C3b molecules is necessary to convert a C3 convertase to a high affinity C5 convertase, the additional C3b molecules play no role in the regulation of C5 convertase by C4BP.