Regulation of complement activation.

CASE 31 Hereditary Angioedema

Regulation of complement activation.

Complement is a system of plasma proteins that participates in a cascade of reactions, generating active components that allow pathogens and immune complexes to be destroyed and eliminated from the body. Complement is part of the innate immune defenses of the body and is also activated via the anti­ bodies produced in an adaptive immune response. Complement activation is generally confined to the surface of pathogens or circulating complexes of antibody bound to antigen.

Complement is normally activated by one of three routes: the classical path­ way, which is triggered by antigen:antibody complexes or antibody bound to the surface of a pathogen; the lectin pathway, which is activated by mannose-binding lectin (MBL) and the ficolins; and the alternative pathway, in which complement is activated spontaneously on the surface of some bacteria. The early part of each pathway is a series of proteolytic cleavage events leading to the generation of a convertase, a serine protease that cleaves complement component C3 and thereby initiates the effector actions of complement. The C3 convertases generated by the three pathways are different, but evolutionarily homologous, enzymes. Complement compon­ ents and activation pathways, and the main effector actions of complement, are summarized in Fig. 31.1.

The principal effector molecule, and a focal point of activation for the system, is C3b, the large cleavage fragment ofC3. If active C3b, or the homologous but less potent C4b, accidentally becomes bound to a host cell surface instead of a pathogen, the cell can be destroyed. This is usually prevented by the rapid hydrolysis of active C3b and C4b if they do not bind immediately to the sur­ face where they were generated. Protection against inappropriate activation of complement is also provided by regulatory proteins.

One of these, and the most potent inhibitor of the classical pathway, is the C1 inhibitor (ClINH) . This belongs to a family of serine protease inhib­ itors (called serpins) that together constitute 20% of all plasma proteins. In addition to being the sole known inhibitor of C1, CIINH contributes to the

I Topics bearing on this case:

Classical pathway of complement activation

Inhibition of C1 activation

Alternative pathway of complement activation

– Inflammatory effects of complement activation

Regulation of C4b IThis case was prepared by Raif Geha, MD, in collaboration vvith Arturo Borzutzkyr, MD.

3 Case 31: Hereditary Angioedema Fig. 31.1 Overview of the main components and effector actions of complement. The early events of all three pathways of complement activation involve a series of cleavage reactions that culminate in the formation of an enzymatic activity called a C3 convertase, which cleaves complement component C3 into C3b and C3a. The production of the C3 convertase is the point at which the three pathways converge and the main effector functions of complement are generated. C3b binds covalently to the bacterial cell membrane and opsonizes the bacteria, enabling phagocytes to internalize them. C3a is a peptide mediator of local inflammation. C5a and C5b are generated by the cleavage of C5b by a C5 convertase formed by C3b bound to the C3 convertase (not shown in this simplified diagram). C5a is also a powerful peptide mediator of inflammation. C5b triggers the late events in which the terminal components of complement assemble into a membrane­ attack complex that can damage the membrane of certain pathogens. Although the classical complement activation pathway was first discovered as an antibody-triggered pathway, it is now known that C 1 q can activate this pathway by binding directly to pathogen surfaces, as well as paralleling the lectin activation pathway by binding to antibody that is itself bound to the pathogen surface. In the lectin pathway, MASP stands for man nose-binding lectin-associated serine protease.

CLASSICAL PATHWAY

Antigen:antibody complexes (pathogen surfaces)

.,,-l 7

C1q, Clr, Cls C4 C2

~

J~

C3a, CSa

.,,-l 7

Peptide mediators of inflammation,

phagocyte recruitment

LECTIN PATHWAYI I Mannose-binding lectin or ficolln binds carbohydrate

on pathogen surfaces

.,,-l 7

MBUficolin, MASP-2 C4 C2

~ 7 C3 convertase

~ l C3b

.,,-l 7

Binds to complement receptors on phagocytes

~ ‘z

Opsonization of pathogens

Removal of immune complexes

I I ALTERNATIVE PATHWAY

Pathogen surfaces

.,,-l 7

C3 B 0

)

~ T”miMIcomplement components CSb C6 C7 C8 C9

.,,-l 7

Membrane-attack complex,

lysis of certain pathogens and cells

regulation of serine proteases of the clotting system and of the kinin system, which is activated by injury to blood vessels and by some bacterial toxins. The main product of the kinin system is bradykinin, which causes vasodilation and increased capillary permeability.

ClINH’intervenes in the first step of the complement pathway, when CI binds to immunoglobulin molecules on the surface of a pathogen or antigen:antihody complex (Fig. 31.2). Binding of two or more of the six tulip-like heads of the Clq component of CI is required to trigger the sequential activation of the two associated serine proteases, Clr and CIs. ClINH inhibits both of these pro­ teases, by presenting them with a so-called bait -site, in the form of an arginine bond that they cleave. !lJhen Clr and CIs attack the bait-site they covalently bind CIINH and dissociate from Clq. By this mechanism, the Cl inhibitor lim­ its the time during which antibody-bound CI can cleave C4 and C2 to generate C4b2a. the classical pathway C3 convertase.

Activation of CI also occurs spontaneously at low levels vvithout binding to an antigen:antibody complex, and can be triggered further by plasmin, a pro­ tease of the clotting system, which is also normally inhibited by Cl1NH. In the absence of ClINH, active components of complement and bradykinin are produced. This is seen in hereditary angioedema (HAE), a disease caused by a genetic defiCiency of C lINH.

Case 31: Hereditary Angioedema ~

o

Cl q binds to IgM on bacterial surface Clq binds to at least two IgG molecules on bacterial surface

r C11NH

o

Binding of Cl q to Ig activates Cl r, which cleaves and activates the serine protease Cl s

Cl lNH dissociates Clr and Cls Irom the active Cl complex

:B C1s r C1 1NH C1r

o o

:BC1S

Fig. 31.2 Activation of t he classical pathway of complement and intervention by C1INH. In the left panel, one molecule of IgM, bent into the ‘staple’ conformation by binding several identical epitopes on a pathogen surface, allows binding by the globular heads of C1 q to its Fc pieces on the surface of the pathogen . In the right panel, multiple molecules of IgG bound to the surface of the pathogen allow binding by C1 q to two or more Fc pieces. In both cases, binding of C 1 q activates the associated C 1 r, whi<;h becomes an active enzyme that cleaves the proenzyme C1 s, a serine protease that initiates the classical complement cascade. Active C1 is inactivated by C1INH, which binds covalently to C1 rand C1 s, causing them to dissociate from the complex. There are in fact two C1 r and two C1 s molecules bound to each C1 q molecule, although for simplicity this is not shown here. It takes four molecules of C11NH to inactivate all the C1 rand C1 s.

The case of Richard Crafton: afailureof communication as well as of complement regulation.

Richard Crafton was a17-year-old hIgh-school senior when he had an attack of severe abdominal pain at the end of a school day. The pain came as frequent sharp spasms and he began to vomit. After 3 hours, the pain became unbearable and he went to the emergency room at the local hospital.

At the hospital, the intern who examined him found no abnormalities other than dry mucous membranes of the mouth, and atender abdomen.There was no point tender­ ness to indicate appendicitis. Richard continued to vomit every 5 minutes and said the pain was getting worse.

A surgeon was summoned. He agreed with the intern that Richard had an acute abdominal condition but was uncertain of the diagnosis. Blood tests showed an elevated red blood cell count, indicating dehydration. The surgeon decided to pro­ ceed with exploratory abdominal surgery. A large midline Incision revealed a moder­ ately swollen and pale jejunum but no other abnormalities were noted. The surgeon removed Richard’s appendix, which was normal, and Richard recovered and returned to school 5 days later.

What Richard had not mentioned to the intern or to the surgeon was that, although he had never had such severe pains as those he was experiencing when he went to the

~ Case 31: Hereditary Angioedema

Fig. 31.3 Hereditary angioedema. Transient localized swelling that occurs in this condition often affects the face.

emergency room, he had had episodes of abdominal pain since he was 14 years old. No one in the emergency room asked him if he was taking any medication, or took a family history or a history of prior illness. If they had, they would have learned that Richard’s mother, his maternal grandmother,and amaternal uncle, also had recurrent episodes of severe abdominal pain, as did his only sibling, a 19-year-old sister.

As a newborn,Richard was prone to severe colic. When he was 4 years old, abump on his head led to abnormal swelling. When he was 7, a blow with a baseball bat caused his entire left forearm to swell to twice its normal size. In both cases, the swelling was not painful, nor was it red or Itchy, and it disappeared after 2days. At age 14 years,he began to complain of abdominal pain every few months, sometimes accompanied by vomiting and,more rarely, by clear, watery diarrhea.

Richard’s mother had taken him at age 4years to an immunologist, who listened to the family history and immediately suspected hereditary angioedema. Thediagnosis was confirmed on measuring key complement components. C11NH levels were 16% of the normal mean and C41evels were markedly decreased, while C3 levels were normal.

When Richard turned up for a routine visit to his immunologist a few weeks after his surgical misadventure, the immunologist, noticing Richard’s large abdominal scar, asked what had happened. When Richard explained, he prescribed daily doses of Winstrol (stanozolol). This caused a marked diminution in the frequency and sever­ ity of Richard’s symptoms. When Richard was 20 years old, purified C11NH became available; he has since been infused intravenously on several occasions to alleviate severe abdominal pain, and once for swelling of his uvula, pharynx, and larynx. The Infusion relieved his symptoms within 25 minutes.

Richard subsequently married and had two children. The C11NH level was found to be normal in both newborns.

Hereditary angioedema.

Individuals like Richard with a hereditary deficiency of ClINH are subject to recurrent episodes of circumscribed swelling of the skin (Fig. 31.3), intes­ tine, and airway. Attacks of subcutaneous or mucosal swelling most com­ monly affect the extremities, but can also involve the face, trunk, genitals, lips, tongue, or larynx. Cutaneous attacks cause temporary disfigurement but are not dangerous. When the swelling occurs in the intestine it causes severe abdominal pain, and obstructs the intestine so that the patient vomits. When the colon is affected, watery diarrhea may occur. Swelling in the larynx is the most dangerous symptom, because the patient can rapidly choke to death. HAE attacks do not usually involve itching or hives, which is useful to differen­ tiate this disease from allergic angioedema. However, a serpiginous, or linear and wavy, rash is sometimes seen before the onset of swelling symptoms. Such episodes may be triggered by trauma, menstrual periods, excessive exercise, exposure to extremes of temperature, mental stress, and some medications such as angiotensin-converting enzyme inhibi tors and oral contraceptives.

HAE is not an allergic disease, and attacks are not mediated by histamine. .HAE attacks are associated with activation of four serine proteases, which are normally inhibited by ClINH. At the top of this cascade is Factor XlI , which directly or indirectly activates the other three (Fig. 31.4) . Factor XlI is normally activated by injury to blood vessels, and initiates the kinin cascade, activating

Case 31: Hereditary Angioedema ~

Fig. 31.4 Pathogenesis of hereditary angioedema. Activation of Factor XII leads to the activation of kallikrein, which cleaves kininogen to produce the vasoactive peptide bradykinin; nalso leads to the activation of plasmin, which in turn activates C1. C1 cleaves C2, whose smaller fragment C2b is further cleaved by plasmin to generate the vasoactive peptide C2 kinin. The red bars represent inhibition by C1INH.

kallikrein, which generates the vasoactive peptide bradykinin. Factor XII also indirectly activates plasmin, which, as mentioned earlier, activates C1 itself. Plasmin also cleaves C2b to generate a vasoactive fragment called C2 kinin. In patients deficient in ClINH, the uninhibited activation of Factor XII leads to the activation of kallikrein and plasmin; kallikrein catalyzes the formation of bradykinin, and plasmin produces C2 kinin. Bradykinin is the main mediator responsible for HAE attacks by causing vasodilation and increasing the per­ meability of the postcapillary venules by causing contraction of endothelial cells so as to create gaps in the blood vessel wall (Fig. 31.5). This is responsible for the edema; movement of fluid from the vascular space into another body compartment, such as the gut, causes the symptoms of dehydration as the vascular volume contracts.

Treatment of HAE can focus on preventing attacks or on resolving acute epi­ sodes. Purified or recombinant ClINH is an effective therapy in both these settings. A kallikrein inhibitor and a bradykinin receptor antagonist have also been developed to target the kinin cascade and bradykinin activity.

Questions.

mActivation of the complement system results in the release of histamine and chemokines, which normally produce pain, heat, and itching. Why is the edema Auid in HAE free of cellular components, and why does the swelling not itch?

QJ Richard has a markedly decreased amount of C4 in his blood. This is because it is being rapidly cleaved by activated C1. What other complement component would you expect to find decreased? Would you expect the alternative pathway components to be low, normal, or elevated? What about the terminal components?

Fig. 31.5 Contraction of endothelial cells creates gaps in the blood vessel wall. A guinea pig was injected intravenously with India ink (a suspension of carbon particles). Immediately thereafter the guinea pig was injected intradermally with a small amount of activated C1 s. An area of angioedema formed about the injected site, which was biopsied 10 minutes later. An electron micrograph reveals that the endothelial cells in post-capillary venules have contracted and formed gaps through which the India ink particles have leaked from the blood vessel. L is the lumen of the blood vessel; P is a polymorphonuclear leukocyte in the lumen; rbc is a red blood cell that has leaked out of the blood vessel. Micrograph courtesy of Kaethe Willms.

Activation of Factor XII

Activation of kallikrein

Cleavage of kininogen to generate bradykinin,

vasoactive peptide

Activation of proactivator