补体蛋白参与非特异性与特异性的防御反应.ppt

1,Biomaterials science 4.4 The Complement System,April 14th 2010 PPT prepared by 丁育民,2,4.4 The Complement System, /ghaffar/complement.htm April 7 2010 PPT prepared by 丁育民,3,Basic mechanisms of innate immunity - phagocytes,1.,2.,3.,4,Basic mechanisms of innate immunity complement activation,1.,2.,3.,4.,5,Functions of complement,6,補體蛋白參與非特異性與特異性的防禦反應,( 一 ) 補體系統是一群約 20 多種不同蛋白質的總稱 , 通常是處於不活化狀態 , 存在於血液的血漿蛋白中 , 惟有受到刺激才會被激活 , 活化是以連鎖反應的方式 , 一旦殷勤了一個因子 , 便會一個接著一個 , 以一定序列敢動開來。 在特異性防禦中 , 補體蛋白其活化的連鎖反應過程最先被確認出 , 所以稱為古典路徑 (classical pathway)。 這個過程最先是由抗原一抗 體複合體的刺激而敢動。 當抗體與入侵病原體的抗原 (如 : 細菌)發生專一性的結合後 , 抗體值定區的某特殊反應部位被活化, 便與補體蛋白分子直接結合 , 此補體蛋白將相鄰抗體分子之間的空隙形 成一橋狀鍵結。 這種抗體與補體的結合會激活補體蛋白, 被活化的補體蛋白便敢動古典路徑的連鎖反應。 過程中, 被活化的補體蛋白 聚集形成細胞膜攻擊複合體 (membrane attack complex) 。 此複合體 能襄入病原菌的雙層脂膜, 使其穿孔導致細胞溶解。作用方式與細胞毒性 T 細胞非常相似, 只是補體蛋白的目標細胞為入侵者本身而非個體細胞。,7,補體蛋白參與非特異性與特異性的防禦反應,( 二 ) 補體系統的另一套活化連鎖反應過程 , 稱為替代路徑 (alternative pathway) 。 它不需要抗體的參與 , 所以在作用的目標上為非專一性的可補體替代路徑亦會生成細胞膜攻擊複合體。 例如 : 多種細菌、 酵母菌、病毒、病毒感染的細胞、原生動物的寄生蟲 (protozoan parasites) 等所合成分可以在沒抗體協助的情況下 , 直接活化補體系統 , 形成細胞膜攻擊複合體。 替代路徑另一個重要結果 , 在於補 體參與發炎反應。 活化的補體蛋白經由與含組織胺的細胞 (histamine- containing cells) 肥胖細胞和嗜鹼性球結合 , 會促使其釋出組織胺。 組織胺相當於化學警報 , 進入局部組織液中 , 警告個體的防禦系統 , 發出一個局部地區受傷的信號 , 使局部血流量增加 , 非特異性 反應提高。 某些補體蛋白也可以吸引吞噬細胞進入感染部位 , 加強 吞噬作用。,8,補體蛋白參與非特異性與特異性的防禦反應,( 三 ) 補體蛋白亦會協同吞噬細胞完成對病原體的破壞 ; 當活化的補體蛋 自黏附在外來的細胞上 , 並刺激吞噬細胞的吞噬作用 , 這個過程稱 為調理作用 (opsonization)( 抗體分子覆蓋在病原體上 , 也具相同效果) 。 個體免疫系統中 , 另一種團隊合作的例子為聯合補體、抗體及吞噬細胞在免疫黏著(immune adherence)現象中共同作用的方式。 當微生物表面覆蓋了抗體與補體蛋白後 ,較易黏著於血管壁的表面 ,使血液循環中的吞噬細胞很容易捕獲此病源體並吞食之。,9,The Complement System,Overview Sometimes the interaction of antibodies with antigen is useful by itself. For example, coating a virus or bacterium thus preventing it from binding to and invading a host cell (e.g., antipolio antibodies); binding to a toxin molecule (e.g., diphtheria or tetanus toxin) thus keeping the toxin from entering a cell where it does its dirty work.,10,But most of the time, the binding of antibodies to antigen performs no useful function until and unless it can activate an effector mechanism. The complement system serves several effector roles. So, the complement system provides the actual protection from the response while the interaction of antibodies and antigen provides the specificity of the response. Put another way, antibodies “finger“ the target, complement destroys it.,11,Features of the system,The complement system consists of some 30 proteins circulating in blood plasma. Most of these are inactive until they are cleaved by a protease which, in turn, converts them into a protease. Thus many components of the system serve as substrate of a prior component and then as an enzyme to activate a subsequent component. This pattern of sequential activation produces an expanding cascade of activity.,12,The Classical Pathway,The binding of antibody to its antigen often triggers the complement system through the so-called classical pathway. It can occur in solution or when the antibodies have bound to antigens on a cell surface.,13,The proteins of the classical pathway,C1 C1 exists in blood serum as a molecular complex containing: 6 molecules of C1q 2 molecules of C1r 2 molecules of C1s,14,The constant regions of mu chains (IgM) and some gamma chains (IgG) contain a binding site for C1q. (A single molecule of IgM is enough to initiate the pathway. IgG is far less efficient, requiring many molecules to do so.),15,Binding of C1q activates C1s and C1r. Activated C1s (a serine protease) cleaves two serum proteins: C4 is cleaved into a large fragment C4b, which binds covalently to sugar residues on cell-surface glycoproteins, and a smaller, inactive, fragment of C4a which diffuses away. C2 is cleaved into C2b, which binds noncovalently to a site on C4b, leaving a smaller, inactive, fragment of C2a which diffuses away. The complex of C4b2b is called “C3 convertase“ because it catalyzes the cleavage of C3. (C4b2b is also a serine protease.),16,C3,C3 is the most abundant protein of the complement system (1.3 mg/ml). Because of its abundance and its ability to activate itself (by a mechanism described below), it greatly magnifies the response.,A hormone, drug or chemical which is capable of eliciting vasoconstrictive or vasodilative action on the vasculature. Examples include histamine and herparin, released at the sites of infection which stimulate vasodilation of arterioles and facilaitate an inflammatory response at the site of infection.,17,C3,C4b2b cuts C3 into two major fragments: C3b, which binds covalently to glycoproteins scattered across the cell surface. Macrophages and neutrophils have receptors for C3b and can bind the C3b-coated cell or particle preparatory to phagocytosis. This effect qualifies C3b as an opsonin. C3a This small fragment is released into the surrounding fluids. It can bind to receptors on basophils and mast cells triggering them to release their vasoactive contents (e.g., histamine). Because of the role of these materials in anaphylaxis全身型過敏性反應, C3a is called an anaphylatoxin.,18,C3,Some of the C3b binds to molecules of C5 creating an allosteric(酵素,與一種分子inhibitor結合後而改變其功能者) change that exposes them to cleavage by C4b2b (which is thus a “C3/C5 convertase“.),19,C5,Cleavage of C5 by the C3/C5 convertase initiates the assembly of a set of complement proteins that make up the membrane attack complex. (The membrane attack complex can also be formed by another C5 convertase produce by the “alternative pathway“,20,The Alternative Pathway,The complement system can also be triggered without antigen-antibody complexes. Even in their absence, there is a spontaneous conversion of C3 to C3b. Ordinarily the C3b is quickly inactivated: the C3b binds to inhibitory proteins and sialic acid唾液酸 present on the surface of the bodys own cells, and the process is aborted. However, bacteria and other foreign materials that may get into the body lack these proteins and have little or no sialic acid. So the C3b binds a protein called Factor B forming a complex of C3bBb. C3bBb is also a C3 convertase acting on more C3 to form: C3bBbC3b, which is a C5 convertase and can start the assembly of the membrane attack complex. more C3b! This second function (shown here) creates a positive feedback loop, amplifying what might have started as a small reaction (the formation of C3b by either or both the classical and alternative pathways) into a massive production of C3b.,21,The Membrane Attack Complex,Cleavage of C5 by the C3/C5 convertase, produces: C5a, which is released into the fluid surroundings is a potent anaphylatoxin (like C3a) is a chemotactic attractant for neutrophils C5b, which serves as the anchor for the assembly of a single molecule each of C6; C7, and C8. The resulting complex C5b678 guides the polymerization of as many as 18 molecules of C9 into a tube inserted into the lipid bilayer of the plasma membrane. This tube forms a channel allowing the passage of ions and small molecules. Water enters the cell by osmosis and the cell lyses.,22,The electron micrograph shows holes punched through the cell wall of the Gram-negative bacterium Shigella志賀桿菌 dysenteriae by the terminal components of the complement system. (Some of the holes are larger than expected for C9 channels and probably were enlarged later by the action of lysozyme.),23,Summary: Effector Functions of Complement,Cell lysis is only one function (and probably not the most important one) of the complement system. The complement system acts in several ways to mobilize defense mechanisms. Opsonization by C3b targets foreign particles for phagocytosis. Chemotaxis by C5a attracts phagocytic cells to the site of damage. This is aided by the increased permeability of the capillary beds mediated by C3a and C5a.,24,Summary: Effector Functions of Complement,The early complement components are also important for solubilizing antigen-antibody complexes assisting in their catabolism and elimination from the body. Failure of this function can lead to immune complex disorders. Lysis of antibody-coated cells. (In some cases, this causes more harm than good; complement-mediated lysis can cause such serious disorders as Rh disease immune hemolytic anemia immune thrombocytopenic purpura Promoting antibody formation. Breakdown of C3b generates a fragment (C3d) that binds to antigens enhancing their uptake by dendritic cells and B cells .,25,Disorders of the Complement System,With so many proteins involved, it is not surprising that inherited deficiencies of one or another are sometimes encountered in humans. Four examples: C3. An inherited deficiency of C3 predisposes the person to frequent bouts of bacterial infections. C2. Curiously, immune complex disorders, not bacterial infections, are the main problem with a deficiency of C2 (or of one of the other “early“ components like C1q, C1r, C1s, or C4). This emphasizes the important role of the complement system in clearing away antigen-antibody complexes. A deficiency of C2 (or one of the other early components) is frequently found in patients with the autoimmune disorder system lupus erythematosus紅斑性狼瘡 (SLE).,26,Disorders of the Complement System,C9. Another curiosity: most people who cannot make C9 have no more of a problem with bacterial infections than those who can. Laboratory studies suggest that the C5b678 complex by itself is able to lyze bacteria although not as efficiently as C9. C1INH. A deficiency of C1INH produces hereditary angioneurotic edema (HANE). Patients are at risk of occasional explosive triggering of the complement system. The massive release of anaphylatoxins (C3a, C5a) may cause dangerous swelling (edema) of the airways, as well as of the skin and intestine.,27,Introduction,Complement refer to plasma components necessary for antibody -mediated bactericidal activity complement is composed of more than 30 distinct plasma and membrane bound proteins involving three separate pathways: classical, alternative, and lectin pathway directly and indirectly contributes both to innate inflammatory reactions and to cellular immune responses.,28,Classical pathway,Classical pathway ( CP ) activated primarily by immune complexes ( ICs ) composed of antigen and specific antibody. The proteins of this pathway are C1, C2, C4, C1- inhibitor ( C1-Inh ), and C4 binding protein ( C4bp ).,29,FIG. 1. Complement activation by the classical pathway (CP).,Upon binding to the Fc (Fragment, crystallizable) region of an immune complex, C1 is activated and cleaves C4, exposing its thioester, which permits covalent attachment of C4b to the activating surface. C2 is cleaved, producing C2b, which binds to C4b to form the CP C3 convertase. C2b is a serine protease that specifically acts on C3 to generate C3b and C3a.,30,FIG. 1. Complement activation by the Lectin pathway,The lectin pathway is also illustrated. MBL(mannan binding lectin) recognizes certain sugar residues (mannose, Nacetylglucosamine) on the surface of an activator (bacteria). MASP-1 appears to activate MASP-2, which then cleaves both C4 and C2 of the CP, generating the CP C3 convertase.,MASP: MBL-associated serine proteases,31,C1 complement,The CP is in a state of continuous low-level activation essentially primed to react vigorously in the presence of an IC. When an IC forms, the cascade is initiated when C1 binds to the Fc (Fragment, crystallizable) portion of an antigen-antibody complex. C1q, C1r, and C1s One end of C1q binds to an IC formed between an antigen and one molecule of immunoglobulin ( Ig ) M or several closely believed to produce spaced IgG molecules .,An Fc receptor is a protein found on the surface of certain cells - including natural killer cells, macrophages, neutrophils, and mast cells - that contribute to the protective functions of the immune system. Its name is derived from its binding specificity for a part of an antibody known as the Fc (Fragment, crystallizable) region.,32,This interaction is believed to produce a conformational change in the C1q results in activation of the two C1r and then the two C1s subunits. The proteolysis of C1s completes the activation of C1 which then proceeds to act on the next proteins in the cascade, C4 and C2. C4 is composed of three separate chains, a,b and , bound together by disulfide bonds.,33,O=CS represents the reactive thioester bond that permits covalent attachment to surface nucleophiles (hydroxyl or amino groups). The pattern of proteolytic degradation and the resulting fragments are also shown. Although factor I is the relevant in vivo protease, some of these same fragments can be generated with trypsin, plasmin, and thrombin.,FIG. 2. Schematic illustration of C4 and C3 protein structures.,34,Upon cleavage of C4, the buried thioester becomes exposed and available to react with a surface containing amino or hydroxyl moieties.,35,About 5% of the C4b molecules produced react through the thioester and become covalently attached to the surface. C1 produces a number of surface-bound C4b sites. The C4b protein, attached to the surface, acts as a receptor for C2 after binding to C4b, C2 becomes a substrate for C1s.,36,The C2b protein is another serine protease in association with C4b, represents the classical pathway C3/C5 convertase.,37,C3b can become attached to the surface surrounding every C4bC2b complex . creating a C3b-C4bC2b complex that acts as a C5 convertase.,38,C5 is also cleaved by C2b to produce fragments designated C5a and C5b. The C5b molecule combines with the proteins of the terminal components to form the membrane attack complex. C5a is a potent inflammatory mediator and is responsible for many of the adverse reactions.,39,Lectin pathway,A protein called mannose (甘露糖) binding protein (or mannan binding lectin, MBL) discovered of a third pathway that leads to complement activation is an acute phase protein, so its concentration in plasma increases substantially during an infection. is similar in structure to C1q, binds the MASP proteins. Upon activation of MBLMASP-1MASP-2, the MASP protease components cleave C4 and C2, forming a CP C3 convertase.,40,41,Alternative pathway,The ability of a yeast cell wall preparation, called zymosan(酵母多糖), to consume C3 without affecting the amount of C1, C2, or C4. A new protein, called properdin implicated in initiating C3 activation independent of the CP called the properdin pathway.,Properdin: 制菌前素,(一種正常血清球蛋白,有補體C3及鎂離子存在時,可殺菌及消滅病毒).,42,43,Their actions can be conceptually divided into three phases: initiation, amplification, and regulation. Initiation is a spontaneous process: nonselective nature of complement the C3 molecules in plasma undergo a conformational change that results in hydrolysis of the thioester group. producing an activated form of C3 called C3 (H2O), that will bind to factor B.,44,45,FIG. 3. Complement activation by the alternative pathway (AP).,The spontaneous conversion of C3 to C3(H2O) permits the continuous production of C3b from C3, a process called C3 tickover. In the presence of an activating surface, the C3b is covalently bound and becomes the focal point for subsequent interactions. The bound C3b is recognized by factor B, which is then cleaved by factor D to produce a surface bound C3 convertase (C3bBb). This results in amplification of the original signal to produce more convertase.,46,Most of the C3b produced is hydrolyzed and inactivated, a process that has been termed“ C3 tickover” ensures a constant supply of reactive C3b molecules to deposit on foreign surfaces. Recognition of the C3b by factor B, cleavage by factor D, and generation of more C3 convertase leads to the amplification phase. Eventually, a C3b molecule attaches to one of the C3 convertase sites.,47,48,FIG. 5. Control of complement activation by factors H, I, and C4 binding protein.,The extent to which complement activation occurs on different surfaces is dependent on the ability of fH or C4BP to recognize C3b or C4b on the surface. Degradation by factor I results in irreversible inactivation and the production of C3 and C4 fragments recognized by various complement receptors on WBC.,49,This C3b-C3bBb complex is the alternative pathway C5 convertase. While the C3bBb convertase is controlled by factors H and l, the surface-bound C3Bb convertase is not. The adsorption of C3 does not occur if the polystyrene surface is precoated with fibrinogen, where many other proteins can compete with C3 for binding to a biomaterial surface.,50,Membrane attack complex,All three pathways lead to a common point : cleavage of C5 to produce C5b and C5a. C5a is a potent inflammatory mediator. C5b initiates the formation of a macromolecular complex of proteins called the membrane attack complex ( MAC ). disrupts the cellular lipid bilayer, leading to cell death,51,FIG. 4. Conversion of C5 produces C5a and leads to formation of the membrane attack complex (MAC). C5a binds to receptors on a variety of cells and results in numerous activities. C5b, formed by the CP, lectin, or the AP, binds C6 and C7 to form a complex that associates with the plasma membrane.,52,FIG. 4. Conversion of C5 produces C5a and leads to formation of the membrane attack complex (MAC). This C5b67 multimer then binds C8, which results in the formation of a small hole in the lipid bilayer that allows small molecules to pass through. Association of multiple C9 proteins enlarges the pore, leading to loss of membrane integrity and cell death.,53,C5b67 has amphiphilic properties allow it to bind to, and partially insert into, lipid bilayers. The C5b678 complex disrupts the plasma membrane and produces small pores ( r 1 nm ) permit leakage of small molecules. C9 bind to the C5b678 complex and insert into the membrane enlarges the pore to about 10 nm and can lead to lysis and cell death. Formation of MAC on host cells results in a number of activation responses (elevated ca2+, arachidonic acid (花生烯酸)metabolism, cytokine production).,5