Session 4 immunity .pdf

The Complement System MB.Ch.B Dr Safeen Othman Mahmood FKBMS Clinical Microbiology – Infectious Diseases Learning objectives At the end of this session students should be able to: • Define complement system • Understand classical, alternative and lectin pathways • Understand role of complement s...

The Complement System MB.Ch.B Dr Safeen Othman Mahmood FKBMS Clinical Microbiology – Infectious Diseases Learning objectives At the end of this session students should be able to: • Define complement system • Understand classical, alternative and lectin pathways • Understand role of complement system in the immune system 2 Complement System Complement system (complement) represents a group of proteins normally found in serum in inactive form, but when activated they augment the immune responses. They are glycoproteins synthesized mainly by hepatocytes, although significant amounts are also produced by blood monocytes, tissue macrophages, and epithelial cells of the gastrointestinal and genitourinary tracts. Complements constitute about 5% of normal serum proteins. Their level does not increase following either infection or vaccination. 3 Complement, General Properties 1. Binding to Fc region of antibody: The effector function of complement is mediated by binding with Fc portion of antibody. The binding of complement to an antibody is described by various terms as, fixing or consumption (as it disappears from serum following binding). 2. Role on antigen: In the classical pathway, complements do not bind to free antibodies but they can only fix to those antibodies which are bound with antigens. However fixation of complement is not influenced by the nature of antigens, but only by the class of antibody. 4 Complement, General Properties 3. Species nonspecific: Complements are present in the sera of all mammals, birds, amphibians and fish. Complements from one species can react with antibodies from other species, though the efficiency decreases with increase in taxonomic distance. 4. Heat labile: Complements get denatured by heating the serum at 56°C for 30 minutes. Such serum with lost complement activity is called inactivated serum. 5 Complement Components Complement system comprises of about 30 serum proteins grouped into complement components, the properdin system and the regulatory proteins. Complement components are named by numerals. There are nine components; C1 to C9. C1 has three subunits- C1q, C1r and C1s. Properdin system and the regulatory proteins are named by letter symbols, e.g., factor-B, Properdin, or regulatory factor H. 6 Complement activation pathways Complement can be activated through three pathways: • Classical pathway- Antibody-dependent pathway. Pathway is triggered by the antigen antibody complex formation. • Alternative pathway- Antibody-independent pathway, triggered by the antigen directly. • Lectin pathway is a recently described pathway. It resembles classical pathway but it is antibody-independent. 7 Complement activation pathways 8 Complement activation All the complement proteins are synthesized in inactive form (e.g. zymogens) and are activated by proteolysis. Complements have two unequal fragments (large and small fragment). The larger fragments are usually designated as ‘b’ (e.g. C3b) and the smaller fragments are designated as ‘a’ (e.g. C3a). An exception is C2a which is larger fragment. During proteolysis, the smaller fragment is removed exposing the active site of the larger fragment. 9 Complement activation The larger fragment participates in the cascade reaction of complement pathway and the smaller fragment diffuses away to mediate other functions. The fragments of complements interact in a definite sequential manner with a cascade like effect, which leads to formation of a complex. Such complex having enzymatic activity is designated by putting a bar over the number or symbol, (e.g. C 3bBb). 10 Stages of complement activation There are four main stages in the activation of any of the complement pathways. 1) Initiation of the pathway 2) Formation of C3 convertase 3) Formation of C5 convertase 4) Formation of membrane attack complex (MAC) All the three pathways differ from each other in their initiation till formation of C3 convertase. Then, the remaining stages are identical in all the pathways. 11 Stages of complement activation 12 Classical Pathway This pathway is antibody dependent, but not all antibodies can bind to complements of classical pathway. In decreasing order of ability of antibodies to fix complement isIgM (most potent) > IgG3> IgG1> IgG2. The other classes of antibodies do not fix complements. CH2 domain on IgG, CH4 on IgM participate in complement binding. The classical pathway begins with activation of C1 and binding to antigen-antibody complex. 13 Classical Pathway 14 Classical Pathway, Initiation The first step is the binding of C1 to the antigen-antibody complex. The first binding portion of C1 is C1q, which reacts with the Fc portion of IgM or IgG bound to antigen. C1q is a hexamer having six globular heads each acting as a combining site. Effective activation of classical pathway begins only when C1q is attached to the Fc portion of antibody by at least two of its globular binding sites. 15 Classical Pathway, Initiation IgM being pentameric, has five Fc regions, hence one molecule of IgM can initiate the pathway. Whereas IgG is monomeric, therefore two IgG molecules are needed to initiate the process. Hence IgM is much efficient stimulator of classical pathway. C1q binding in the presence of calcium ions, in turn activates sequentially C1r followed by C1s. 16 Classical Pathway, Formation of C3 convertase Activated C1s acts as an esterase (C1s esterase), which can cleave C4 to produce C4a (an anaphylatoxin), and C4b which binds to C1 and participates further in complement cascade. C14b in the presence of magnesium ions cleaves C2 into C2a, which remains linked to complement complex, and C2b (has kinin like activity), which is released outside. C14b2a is referred to as C3 convertase of the classical pathway. 17 Classical Pathway, Initiation, Formation of C5 convertase C3 convertase hydrolyses many C3 molecules into two fragments: C3a (an anaphylatoxin) C3b which remains attached to C14b2a to form C14b2a3b complex which acts as C5 convertase of classical pathway. 18 Classical Pathway, Formation of MAC Begins with C5 convertase cleaving C5 into C5a (an anaphylatoxin, released into the medium) and C5b, which continues with the cascade. C5b is extremely labile, gets stabilized by binding soon with C6 and C7 to form C5b67 followed by addition of C8. Hydrophobic regions on C7 and C8 help in penetration into the target cell membrane. This inserted membrane complex (C5b678) has a catalytic property to bind to C9 molecule and then it polymerizes the C9 into a tubular channel of 10 nm diameter 19 Classical Pathway, Formation of MAC Penetration of C9 channels or pores on the target cell membrane. Each tubular channel-hydrophobic outside, hydrophilic inside- free passage of ions and water into the cell-cellular swelling-lysis. C5b6789 destroys the target cell by attacking the cell membrane (MAC) Process of cytolysis is referred to as complement-mediated cytotoxicity. 20 Classical Pathway, Formation of MAC 21 Alternative Pathway Independent of antibody; hence is considered as a part of innate immunity. It differs from the classical pathway in first two stages. It requires three complement proteins present in serum named factor B, factor D and Properdin. 22 Alternative Pathway, initiation Alternative pathway is activated by: • Antigens from pathogen: such as Gram-negative endotoxin or LPS, teichoic acid from Gram-positive bacteria, fungal or yeast cells, trypanosomes, or virus infected cells. • Non microbial initiators: such as human antibodies in complexes- IgA, IgD, tumor cells, cobra venom factor, heterologous RBCs (mouse, rabbit and chicken), anion polymer like dextran sulphate, pure carbohydrates like agar, inulin. 23 Alternative Pathway, initiation First complement component to be involved in alternative pathway is free C3 in the serum. C3 hydrolyzes spontaneously, to generate C3a which diffuses out and C3b fragment which attaches to foreign cell surface antigen. 24 Alternative Pathway, Formation of C3 convertase Factor B binds to C3b coated foreign cells. Factor D acts on factor B, and cleaves it into Ba (diffuses out) and Bb (remains attached). C3bBb has a very short half-life of 5 minutes. Stabilized by properdin (half-life is increased to 30 minutes). 25 Alternative Pathway The last two steps (formation of C5 convertase and formation of MAC) is identical to the classical pathway. 26 Lectin Pathway Complement pathway of innate immunity-works independent of antibody. Mediated through lectin proteins of the host that interact with mannose residues present on microbial surface. Lectin pathway involves all complement components used for classical pathways except C1. Instead of C1, host lectin protein called mannose binding lectins mediate the first ‘initiation’ stage. 27 Lectin Pathway, initiation Activation - Mannose carbohydrate residues of glycoproteins present on microbial surfaces. Mannose binding lectins (MBL) bind to mannose residues on microbial surface. MBL is an acute phase reactant protein, similar to C1q in structure. 28 Lectin Pathway, Initiation After binding of MBL to microbial surface, another host protein called MASP (MBL associated serine protease) gets complexed with MBL. MASP is similar or C1r and C1s and mimics their functions. MBL-MASP complex cleaves C4 which in turn splits C2 and the MBL/MASP-C4b2a acts as C3 convertase. 29 Lectin Pathway The last two steps (formation of C5 convertase and formation of MAC) is identical to the classical pathway. 30 Differences between the complement pathways Classical pathway Alternative pathway Lectin pathway Activator (initiator) Antigen antibody complex Endotoxin IgA, IgD, Cobra venom, Nephritic factor Carbohydrate residue of bacterial cell wall (mannose binding protein) that binds to host lectin antigen. 1st complement activated C3 convertase C5 convertase (C3 convertase + 3b) C1 C3b C4 C14b2a C14b2a3b C3bBb C3bBb3b MBL/MASP-C4b2a MBL/MASP-C4b2a3b Complement level in the serum after activation Immunity All C1-C9: Low C1,C4,C2- Normal Others- Low C1- Normal Others- Low Acquired Innate Innate 31 Effector Functions of Complement MAC and other complement by-products (C3a, C3b, C5a) produced during the activation augment the immune response in many ways. • Target cell lysis by MAC • Inflammatory response • Opsonization • Removing the immune complexes from blood• Viral neutralization 32 Target cell lysis by MAC, complement mediated cell lysis MAC makes pores or channels in the target cell membrane. Allows the free passage of various ions and water into the cell leading to cell swelling, lysis and death. MAC form pores on bacteria, enveloped viruses, damaged cells, tumor cells, etc. 33 Inflammatory response C3a, C4a and C5a serve as anaphylatoxins. They bind to surface receptors of mast cells and induce their degranulation leading to release of histamine and other inflammatory mediators. This increase vascular permeability. 34 Opsonization C3b and C4b -major opsonin- coat the immune complexes and particulate antigens. Phagocytic cells express complement receptors (CR1, CR3 and CR4) for complement components (C3b, C4b). Bind to complement coated antigens and enhance phagocytosis. C5a enhances the CR1 expression on phagocytes by 10 folds. 35 Removing the immune complexes from blood C3b has another role. C3b bound immune complexes is recognized by complement receptor CR1present on RBCs. Immune complexes bound to RBCs are taken to liver and spleen where they are phagocytosed after being separated from the RBCs. 36 Viral neutralization Complements coated on virus surfaces neutralize the viral infectivity by blocking their attachment sites. C3b mediates opsonization of viral particles. Lysis of the enveloped viruses by activation of classical pathway (most viruses) or alternative or lectin pathways (viruses like Epstein Barr virus, rubella etc.) 37 Regulation of Complement Pathways Activation of the complement cascade and the stability of active complement proteins are tightly regulated to prevent complement activation on normal host cells and to limit the duration of complement activation even on microbial cells and antigen-antibody complexes. There are several regulatory factors that checks the complement system in several steps or products. 38 Regulation Of Complement Pathways Regulatory proteins Pathway affected Type of protein Regulatory function DAF (Decay accelerating factor) or CD55 All three pathways Membrane bound Accelerates dissociation of C3 convertase Factor-I All three pathways Soluble Cleaves C4b or C3b by using C4b-binding protein Soluble Binds soluble C5b67 and prevents its insertion into cell membrane Membrane bound Inhibit MAC formation by blocking C9 binding Membrane bound Inhibit MAC formation by blocking C9 binding 1. C3 convertase regulators 2. MAC formation regulators S protein Membrane inhibitor of reactive lysis (MIRL or CD59) Homologous restriction factor All three pathways 39 Complement Deficiencies Many complement deficiencies are associated with increased susceptibility to bacterial infections and/or immune-complex diseases. Complement protein deficiencies C1, C2, C3, C4 Properdin, Factor D Membrane attack complex (C5-C9) Pathway(s) involved Disease/pathology C1, C2, C4-Classical pathway C3- Common deficiency Alternative pathway SLE, glomerulonephritis & pyogenic infections Common deficiency Disseminated Neisseria infection Neisseria and pyogenic infection 40 Complement Deficiencies Complement regulatory protein deficiencies Deficient component Pathway(s) involved Diseases C1 esterase inhibitor Overactive classical pathway Hereditary angioneurotic edema DAF (Decay accelerating factor) & CD59 Factor I Deregulated C3 convertase Increased RBC lysis PNH (Paroxysmal nocturnal hemoglobinurea) Deregulated classical pathway with over consumption of C3 Deregulated alternative pathway with increased C3 convertase activity Immune complex disease; recurrent pyogenic infections Immune complex disease; pyogenic infection Factor H 41 Learning Objectives By the end of this session, students should be able to: 1. Extract the importance of the complement cascade in the prevention of infections. 2. Relate C1 inhibitor (C1INH) deficiency and angioedema. 3. Interpret the importance of the opsonins. 4. Summarize events in the activation of the classical complement cascade. onlyinIgM If IgA Case Study 1: An 18-year-old female college student is hospitalized because of high fever, headache, and skin rash. She has had these symptoms for the past 24 hours. She has a history of recurrent infections, including bacterial meningitis 6 months ago that was success fully treated with ceftriaxone. She takes no medication at home. She denies recent travel and insect bites. She does not use tobacco, alcohol, or illicit drugs. Her temperature is 39.1° C, blood pressure is 104170 mm Hg, and pulse is 110/min and regular. General physical examination reveals a petechial rash on the trunk and extremities, including the palms and soles. Neurologic examination shows lethargy, alertness, fluent speech, and ability to follow commands. She has meningitis evidence of neck stiffness and photophobia. Which of the following immune system impairments is the most likely explanation for her symptoms? Explain and discuss your answer. A. Pure T-cell dysfunction B. Ineffective intracellular killing C. Insufficient lgA production D. Inability to form the membrane attack complex withoutthe MAC therewill benotlysis ofmicroorganisms Answer: D. Inability to form the membrane attack complex Patients with deficiencies of the complement factors that form the membrane attack complex (MAC) (i.e., C5b--C9) often experience recurrent infections by Neisseria species. The MAC is the end-product of antibody complement fixation. It forms a pore in the bacterial cell membrane, leading to electrolyte disturbances, inflow of free water, and eventual cell lysis. N. meningitidis is a common cause of bacterial meningitis, especially in the college dormitory setting. Clinically, N. meningitidis presents with high fever, chills, altered mentation, petechial skin rash from Neisseria induced small vessel vasculitis (especially affecting palms and soles), and ultimately septic shock. The treatment is intravenous ceftriaxone for at least 2 weeks. Educational Objective: Deficiency of the complement factors that form the membrane attack complex (i.e., C5b-C9) results in recurrent infections by Neisseria species. to Case Study 2: A 21-year-old woman comes to the office due to recurrent episodes of selflimited, colicky abdominal pain. She also had an episode of facial swelling that resolved spontaneously. The patient has no other significant past medical history and takes no medications. Examination is unremarkable. Evaluation shows that her complement protein C1, even when not attached to an antigen-antibody complex, is excessively cleaving C2 and C4. Which of the following is most likely increased in this patient? Explain and discuss your answer. A. Antinuclear antibody titer B. Antistreptolysin O titer C. Bradykinin vasodilator D. Free hemoglobin E. Renin ispartofrenin angiotensin ANA disease autoimmune for Rhomboiddiseaseand diagnosis group a ASO titer for streptococcus Answer: C. Bradykinin willinhibitbradykinin c onvertingenzyme angiotensin This patient with recurrent episodes of abdominal pain and an episode of facial swelling likely has angioedema due to C1 inhibitor (C1INH) deficiency. C1INH prevents C1-mediated cleavage of C2 and C4, thereby limiting activation of the complement cascade. It also blocks kallikrein-induced conversion of kininogen to bradykinin, a potent vasodilator that also causes increased vascular permeability. type Acquired or hereditary C1INH deficiency (due to the complete absence of C1INH or the presence of a dysfunctional variant or an anti-C1INH antibody) leads to elevated levels of bradykinin, and patients can develop bradykininassociated angioedema. Symptoms include facial swelling (without urticaria), life-threatening laryngeal edema, and gastrointestinal manifestations (e.g., nausea/vomiting, colicky pain, diarrhea). Management of acute attacks involves supportive care and the administration of C1INH concentrate or a kallikrein inhibitor. Educational Objective: C1 inhibitor (C1INH) deficiency causes increased cleavage of C2 and C4 and results in inappropriate activation of the complement cascade. C1INH also blocks kallikrein-induced conversion of kininogen to bradykinin, a potent vasodilator associated with angioedema. __ itching ACE willinhibit bradykinin Case Study 3: A 3-year-old girl is brought to the office after developing fever and a sore throat. The patient recently entered day care, and similar symptoms have been reported in several of the other children. Physical examination shows exudative pharyngitis and enlarged anterior cervical lymph nodes. A rapid antigen detection test confirms the diagnosis of streptococcal throat infection. Her condition resolves with antibiotic therapy. groupAstreptococcus Several weeks later, she is re-exposed to Streptococcus pyogenes. The bacteria penetrating beyond the surface epithelium are immediately coated with preformed lgG antibodies. Which of the following substances acts in the most similar manner to lgG antibodies to facilitate phagocytosis? Explain and discuss your answer. A. 5-Hydroxyicosatetraenoic acid B. Complement C3b C. Complement C5a D. lmmunoglobulin M E. Leukotriene B4 F L-selectin Answer: B. Complement C3b bephagocytosis withoutopsonizationtherewillnot Opsonization occurs when host proteins such as immunoglobulins or complement bind to the surface of foreign cells (e.g., bacteria) to promote phagocyt osis . The most important opsonins (coating proteins) are immunoglobulin (lg) G and complement C3b, but mannose-binding lectin and C-reactive protein can also opsonize cells. After opsonins are bound to the cell surface, they act as a handle for receptors on phagocytes (e.g., Fc receptors, C3b receptors) to grasp, allowing the phagocytes to more easily engulf the foreign cell. The importance of C3 is demonstrated by the fact that all 3 complement pathways (lectin, classical, and alternative) converge on it, resulting in cleavage into C3a and C3b. C3a helps recruit phagocytic cells and induce inflammation. C3b, in addition to acting as an opsonin, can bind to C3b convertase and form C5 convertase, ultimately triggering the membrane attack complex. Educational Objective: Opsonization occurs when host proteins such as immunoglobulins or e complement bind to foreign cells such as bacteria and coat the surface, enhancing phagocytosis. The most important opsonins (coating proteins) are immunoglobulin G and complement C3b. Case Study 4: lgG autoantibodies against a RBC antigen that are found in a 34-year-old Caucasian female are able to cause cell lysis after binding the C1 complement component. Which of the following is the complement binding site for the immunoglobulin molecule shown below? Explain and discuss your answer. A. A B. B C. C D. D E. E Answer: D. D Both lgG antibodies and lgM antibodies are capable of, and essential for, triggering the classical complement pathway after binding a C1 molecule. The classical pathway would not be able to proceed in the absence of either lgM or lgG. C1 is the complement component that when activated is able to release the catalytic factors responsible for the next steps in the classical complement pathway. In order to be activated, C1 must bind the Fc portions of two different antibodies at specific C1 binding sites. Because lgM circulates in pentameric form (five lgM molecules joined together at their Fc regions by a J chain peptide), it is much more effective in initiating the complement cascade than lgG which circulates in monomeric form (a single circulating immunoglobulin as pictured above). The complement binding site on both lgG and lgM is located in the Fc portion closer to the hinge region. Activation of complement by lgM prior to antigen binding is prevented due to the fact that the C1 binding site on lgM is hidden while unbound lgM is circulating in its planar form. A conformational change in the lgM molecule after antigen binding results in exposure of the C1 binding site. Educational Objective: The classical complement cascade begins with binding of the C1 complement component to either two molecules of lgG or to two molecules of lgM. Because lgM circulates in pentameric form, it is a much better activator of the complement system. The C1 molecule binds to the Fc region of the heavy immunoglobulin chain in the region near the hinge point. 0cg 2instantregion region