Effector cells engage their targets using Fc and C3 receptors

In type II hypersensitivity, antibody directed against cell surface or tissue antigens interacts with the Fc receptors (FcR) on a variety of effector cells and can activate complement to bring about damage to the target cells (Fig. 24.1).

Fig. 24.1 Antibody-dependent cytotoxicity

Effector cells – K cells, platelets, neutrophils, eosinophils, and cells of the mononuclear phagocyte series – all have receptors for Fc, which they use to engage antibody bound to target tissues. Activation of complement C3 can generate complement-mediated lytic damage to target cells directly, and also allows phagocytic cells to bind to their targets via C3b, C3bi, or C3d, which also activate the cells. (MAC, membrane attack complex.)

Once the antibody has attached itself to the surface of the cell or tissue, it can bind and activate complement component C1, with the following consequences:

Effector cells – in this case macrophages, neutrophils, eosinophils, and NK cells – bind to either:

The mechanisms by which these antibodies trigger cytotoxic reactions in vivo have been investigated in FcR-deficient mice. Anti-red blood cell antibodies trigger erythrophagocytosis of IgG-opsonized red blood cells in an FcR-dependent manner. Fc receptor γ chain-deficient mice were protected from the pathogenic effect of these antibodies whereas complement-deficient mice were indistinguishable from wild-type animals in their ability to clear the targeted red cells.

Cells damage targets by releasing their normal immune effector molecules

The mechanisms by which neutrophils and macrophages damage target cells in type II hypersensitivity reactions reflect their normal methods of dealing with infectious pathogens (Fig. 24.2).

Fig. 24.2 Damage mechanisms

Neutrophil-mediated damage is a reflection of normal antibacterial action. (1) Neutrophils engage microbes with their Fc and C3 receptors. (2) The microbe is then phagocytosed and destroyed as lysosomes fuse to form the phagolysosome (3). In type II hypersensitivity reactions, individual host cells coated with antibody may be similarly phagocytosed, but where the target is large, for example a basement membrane (I), the neutrophils are frustrated in their attempt at phagocytosis (II). They exocytose their lysosomal contents, causing damage to cells in the vicinity (III).

Normally pathogens would be internalized and then subjected to a barrage of microbicidal systems including defensins, reactive oxygen and nitrogen metabolites, hypohalites, enzymes, altered pH, and other agents that interfere with metabolism (see Chapters 7 and 14).

If the target is too large to be phagocytosed, the granule and lysosome contents are released in apposition to the sensitized target in a process referred to as exocytosis. Cross-linking of the Fc and C3 receptors during this process causes activation of the phagocyte with production of reactive oxygen intermediates, as well as activation of phospholipase A2 with consequent release of arachidonic acid from membrane phospholipids.

In some situations, such as the eosinophil reaction against schistosomes (see Chapter 15), exocytosis of granule contents is normal and beneficial. However, when the target is host tissue that has been sensitized by antibody, the result is damaging (Fig. 24.3).

Fig. 24.3 Phagocytes attacking a basement membrane

This electron micrograph shows a neutrophil (N) and three monocytes (M) binding to the capillary basement membrane (B) in the kidney of a rabbit containing anti-basement membrane antibody. × 3500. (P, podocyte.)

(Courtesy of Professor GA Andres.)

Antibodies may also mediate hypersensitivity by NK cells. In this case, however, the nature of the target, and whether it can inhibit the NK cells’ cytotoxic actions, are as important as the presence of the sensitizing antibody.

The resistance of a target cell to damage varies. Susceptibility depends on:

For example, an erythrocyte may be lysed by a single active C5 convertase site, whereas it takes many such sites to destroy most nucleated cells – their ion-pumping capacity and ability to maintain membrane integrity with anti-complementary defenses is so much greater.

We now examine some instances where type II hypersensitivity reactions are thought to be of prime importance in causing target cell destruction or immunopathological damage.

Transfusion reactions occur when a recipient has antibodies against donor erythrocytes

More than 20 blood group systems, generating over 200 genetic variants of erythrocyte antigens, have been identified in humans.

A blood group system consists of a gene locus that specifies an antigen on the surface of blood cells (usually, but not always, erythrocytes).

Within each system there may be two or more phenotypes. In the ABO system, for example, there are four phenotypes (A, B, AB, and O), and therefore four possible blood groups.

An individual with a particular blood group can recognize erythrocytes carrying allogeneic (non-self) blood group antigens, and will produce antibodies against them. However, for some blood group antigens such antibodies can also be produced ‘naturally’ (i.e. without previous sensitization by foreign erythrocytes).

Some blood group systems (e.g. ABO and Rhesus) are characterized by antigens that are relatively strong immunogens; such antigens are more likely to induce antibodies.

When planning a blood transfusion, it is important to ensure that donor and recipient blood types are compatible with respect to these major blood groups, otherwise transfusion reactions will occur.

Some major human blood groups are listed in Figure 24.4.

Fig. 24.4 Five major blood group systems involved in transfusion reactions

Not all blood groups are equally antigenic in transfusion reactions – thus RhD evokes a stronger reaction in an incompatible recipient than the other Rhesus antigens; and Fy^a is stronger than Fy^b. Frequencies stated are for Caucasian populations – other races have different gene frequencies.

The ABO blood group system is of primary importance

The epitopes of the ABO blood group system occur on many cell types in addition to erythrocytes and are located on the carbohydrate units of glycoproteins. The structure of these carbohydrates, and of those determining the related Lewis blood group system, is determined by genes coding for enzymes that transfer terminal sugars to a carbohydrate backbone (Fig. 24.5).

Fig. 24.5 ABO blood group antigens

The diagram shows how the ABO blood groups are constructed. The enzyme produced by the H gene attaches a fucose residue (Fuc) to the terminal galactose (Gal) of the precursor oligosaccharide. Individuals possessing the A gene now attach N-acetylgalactosamine (NAGA) to this galactose residue, whereas those with the B gene attach another galactose, producing A and B antigens, respectively. People with both genes make some of each. The table indicates the genotypes and antigens of the ABO system. Most people naturally make antibodies to the antigens they lack. (NAG, N-acetylglucosamine.)

Most individuals develop antibodies to allogeneic specificities of the ABO system without previous sensitization by foreign erythrocytes. This sensitization occurs through contact with identical epitopes, coincidentally expressed on a wide variety of microorganisms.

Antibodies to ABO antigens are therefore extremely common, making it particularly important to match donor blood to the recipient for this system. However, all people are tolerant to the O antigen, so O individuals are universal donors with respect to the ABO system.

Transfusion reactions can be caused by minor blood groups

A number of minor antigens can also induce transfusion reactions, but there is generally only a signficant risk if the person has been sensitized by previous blood transfusions.

MN system epitopes are expressed on the N-terminal glycosylated region of glycophorin A, a glycoprotein present on the erythrocyte surface. Antigenicity is determined by polymorphisms at amino acids 1 and 5.

The related Ss system antigens are carried on glycophorin B.

Proteins expressed on erythrocytes that display allelic variation can also act as blood group antigens. Examples of these include:

• the Kell antigen, a zinc endopeptidase;

• the Duffy antigen receptor for chemokines (DARC); and

• variants of decay accelerating factor (DAF).

The relationship of the blood groups to erythrocyte surface proteins is listed in Figure 24.w1.

Fig. 24.w1 Erythrocyte blood group antigens

Note that blood group epitopes based on carbohydrate moieties, such as ABO and Ii (expressed on the precursor of the ABO polysaccharide), can appear on many different proteins, including Rhesus antigens. Antigens such as Rhesus and Duffy are proteins, so the epitope appears on only one type of molecule. In general, the most important blood group antigens are present at high levels on the erythrocytes, thus providing plenty of targets for complement-mediated lysis or Fc receptor-mediated clearance. (DAF, decay accelerating factor; DARC, Duffy antigen receptor for chemokines.)

Transfusion reactions caused by the minor blood groups are relatively rare unless repeated transfusions are given. The risks are greatly reduced by accurately cross-matching the donor blood to that of the recipient.

Cross-matching ensures that a recipient does not have antibodies against donor erythrocytes

The aim of cross-matching is to ensure that the blood of a recipient does not contain antibodies that will be able to react with and destroy transfused (donor) erythrocytes. For example:

• antibodies to ABO system antigens cause incompatible cells to agglutinate in a clearly visible reaction;

• minor blood group systems cause weaker reactions that may only be detectable by an indirect Coombs’ test (see Fig. 24.9).

Fig. 24.9 Direct antiglobulin test

This test, also called a Coombs’ test, is used to detect antibody on a patient’s erythrocytes. If antibody is present the erythrocytes can be agglutinated by anti-human immunoglobulin. If no antibody is present on the red cells, they are not agglutinated by anti-human immunoglobulin.

If the individual is transfused with whole blood, it is also necessary to check that the donor’s serum does not contain antibodies against the recipient’s erythrocytes. However, transfusion of whole blood is unusual – most blood donations are separated into cellular and serum fractions, to be used individually.

HDNB is due to maternal IgG reacting against the child’s erythrocytes in utero

Hemolytic disease of the newborn (HDNB) occurs when the mother has been sensitized to antigens on the infant’s erythrocytes and makes IgG antibodies to these antigens. These antibodies cross the placenta and react with the fetal erythrocytes, causing their destruction (Figs 24.6 and 24.7). Rhesus D (RhD) is the most commonly involved antigen.

Fig. 24.6 Hemolytic disease of the newborn

Erythrocytes from a RhD⁺ fetus leak into the maternal circulation, usually during birth. This stimulates the production of anti-Rh antibody of the IgG class postpartum. During subsequent pregnancies, IgG antibodies are transferred across the placenta into the fetal circulation (IgM cannot cross the placenta). If the fetus is again incompatible, the antibodies cause erythrocyte destruction.

Fig. 24.7 A child with hemolytic disease of the newborn

There is considerable enlargement of the liver and spleen associated with erythrocyte destruction caused by maternal anti-erythrocyte antibody in the fetal circulation. The child had elevated bilirubin (breakdown product of hemoglobin). The facial petechial hemorrhaging was due to impaired platelet function. The most commonly involved antigen is RhD.

(Courtesy of Dr K Sloper.)

A risk of HDNB arises when a Rh⁺-sensitized Rh^– mother carries a second Rh⁺ infant. Sensitization of the Rh^– mother to the Rh⁺ erythrocytes usually occurs during the birth of the first Rh⁺ infant, when some fetal erythrocytes leak back across the placenta into the maternal circulation and are recognized by the maternal immune system. The first incompatible child is therefore usually unaffected, whereas subsequent children have an increasing risk of being affected, as the mother is resensitized with each successive pregnancy.

Reactions to other blood groups may also cause HDNB, the second most common being the Kell system K antigen. Reactions due to anti-K are much less common than reactions due to RhD because of the relatively low frequency (9%) and weaker antigenicity of the K antigen.

The risk of HDNB due to Rhesus incompatibility is known to be reduced if the father is of a different ABO group to the mother. This observation led to the idea that these Rh^– mothers were destroying Rh⁺ cells more rapidly because they were also ABO incompatible. Consequently, fetal Rh⁺ erythrocytes would not be available to sensitize the maternal immune system to RhD antigen.

This notion led to the development of Rhesus prophylaxis – preformed anti-RhD antibodies are given to Rh^– mothers immediately after delivery of Rh⁺ infants, with the aim of destroying fetal Rh⁺ erythrocytes before they can cause Rh⁻ sensitization. This practice has successfully reduced the incidence of HDNB due to Rhesus incompatibility (Fig. 24.8). Although the number of cases of HDNB has fallen dramatically and progressively, the proportion of cases caused by other blood groups, including Kell and the ABO system, has increased.

Fig. 24.8 Rhesus prophylaxis

(1) Without prophylaxis, Rh⁺ erythrocytes leak into the circulation of a Rh^– mother and sensitize her to the Rh antigen(s). (2) If anti-Rh antibody (anti-D) is injected immediately postpartum it eliminates the Rh⁺ erythrocytes and prevents sensitization. The incidence of deaths due to HDNB fell during the period 1950–1966 with improved patient care. The decline in the disease was accelerated by the advent of Rhesus prophylaxis in 1969.

Autoimmune hemolytic anemias arise spontaneously or may be induced by drugs

Reactions to blood group antigens also occur spontaneously in the autoimmune hemolytic anemias, in which patients produce antibodies to their own erythrocytes.

Autoimmune hemolytic anemia is suspected if a patient gives a positive result on a direct antiglobulin test (Fig. 24.9), which identifies antibodies present on the patient’s erythrocytes. These are usually antibodies directed towards erythrocyte antigens, or immune complexes adsorbed onto the erythrocyte surface.

The direct antiglobulin test is also used to detect antibodies on red cells in mismatched transfusions, and in HDNB.

Autoimmune hemolytic anemias can be divided into three types, depending upon whether they are caused by:

Drug-induced reactions to blood components occur in three different ways

Drugs (or their metabolites) can provoke hypersensitivity reactions against blood cells, including erythrocytes and platelets. This can occur in three different ways (Fig. 24.10).

• The drug binds to the blood cells and antibodies are produced against the drug. In this case it is necessary for both the drug and the antibody to be present to produce the reaction. This phenomenon was first recorded by Ackroyd, who noted thrombocytopenic purpura (destruction of platelets leading to purpuric rash) following administration of the drug sedormid. Hemolytic anemias have been reported following the administration of a wide variety of drugs, including penicillin, quinine and sulfonamides. All of these conditions are rare.

• Drug–antibody immune complexes are adsorbed onto the erythrocyte cell membrane. When drug–antibody immune complexes are adsorbed on to the erythrocyte cell membrane damage occurs by complement-mediated lysis.

• The drug induces an allergic reaction. The drug induces an allergic reaction and autoantibodies are directed against the erythrocyte antigens themselves, as occurs in 0.3% of patients given α-methyldopa. The antibodies produced are similar to those in patients with warm-reactive antibody. However, the condition remits shortly after the cessation of drug treatment.

Fig. 24.10 Drug-induced reactions to blood cells

Three ways that drug treatment can cause damage are illustrated. (1) The drug adsorbs to cell membranes. Antibodies to the drug bind to the cell and complement-mediated lysis occurs. (2) Immune complexes of drugs and antibody become adsorbed to the red cell. This could be mediated by an Fc receptor, but is more probably via the C3b receptor CR1. Damage occurs by complement-mediated lysis. (3) Drugs, presumably adsorbed onto cell membranes, induce a breakdown of self tolerance, possibly by stimulating TH cells. This leads to the formation of antibodies to other blood group antigens on the cell surface. Note that in examples 1 and 2 the drug must be present for cell damage to occur, whereas in 3 the cells are destroyed whether they carry adsorbed drug or not.

Autoantibodies to platelets may cause thrombocytopenia

Autoantibodies to platelets are seen in up to 70% of cases of idiopathic thrombocytopenic purpura (ITP), a disorder in which there is accelerated removal of platelets from the circulation, mediated primarily by splenic macrophages. The mechanism of removal is via the immune adherence receptors on these cells.

ITP most often develops after bacterial or viral infections, but may also be associated with autoimmune diseases including SLE. In SLE, antibodies to cardiolipin, which is present on platelets, can sometimes be detected. Autoantibodies to cardiolipin and other phospholipids can inhibit one aspect of blood clotting (lupus anticoagulant) and can be associated, in some cases, with venous thrombosis and recurrent abortions.

Thrombocytopenia may also be induced by drugs by similar mechanisms to those outlined in Figure 24.10.

Reactions against neutrophils can occur in several autoimmune diseases

Autoantibodies to neutrophil cytoplasmic antigens (ANCAs) are associated with a number of diseases. For example:

Other granule components may also act as antigens in SLE, but less commonly than myeloperoxidase. Such autoantigens are generally neutrophil specific and antibodies can be detected by immunofluorescent staining (Fig. 24.w2). (By contrast, antibodies to MHC antigens, which are also seen in SLE, are highly non-tissue specific.)

Fig. 24.w2 Antibodies to neutrophils

Antibodies to neutrophils are demonstrated by immunofluorescence. In this instance, normal neutrophils were stained with antibodies from a neonate suffering from alloimmune neonatal neutropenia.

p-ANCAs are particularly characteristic of small vessel vasculitis and glomerulonephritis. One of the antigens PR3 that is recognized by ANCA antibodies is also found on endothelium. It has been suggested therefore that the damage could be due to direct action against the blood-vessel wall or indirectly via release of the neutrophils’ granule contents. In Wegener’s granulomatosis the ANCA-antibody titres relate to disease severity. However, their contribution to disease pathogenesis appears to be relatively small, although they do have some diagnostic use.

Antibodies against basement membranes produce nephritis in Goodpasture’s syndrome

A number of patients with nephritis are found to have antibodies to collagen type IV, which is a major component of basement membranes. Collagen type IV undergoes alternate RNA splicing, which produces a number of variant proteins (Goodpasture antigen), but the antibodies appear to bind just those forms that retain the characteristic N terminus. The antibody is usually IgG and, in at least 50% of patients, it appears to fix complement.

Goodpasture’s syndrome usually results in severe necrosis of the glomerulus, with fibrin deposition. The association of this type of nephritis with lung hemorrhage was originally noticed by Goodpasture (hence Goodpasture’s syndrome). Although the lung symptoms do not occur in all patients, the association of lung and kidney damage is due to cross-reactive autoantigens in the basement membranes of the two tissues.

Pemphigus is caused by autoantibodies to an intercellular adhesion molecule

Pemphigus vulgaris is a serious blistering disease of the skin and mucous membranes. Patients have autoantibodies against desmoglein-1 and desmoglein-3, components of desmosomes, which form junctions between epidermal cells (Fig. 24.11). The antibodies disrupt cellular adhesion, leading to breakdown of the epidermis with separation of the superficial layers to form blisters.

Fig. 24.11 Autoantibodies in pemphigus

The antibodies in pemphigus bind to components of the desmosome involved in cell adhesion. Desmoglein-1 and desmoglein-3 are most commonly involved, but other molecules, including the plakins and desmocollin, may also act as autoantigens. Immunofluorescence of human skin stained with anti-IgA.

(Courtesy of Dr R Mirakian and Mr P Collins.)

Clinical disease profiles can be related to the specificity of the antibodies. For example:

The disease profile is also partly dependent on the isotype of the antibodies produced; some patients show strong deposition of IgA (see Fig. 24.11) whereas other have particularly high titres of IgG4. Recently, antibodies to mitochondrial components have also been implicated in the pathology, by inducing apoptosis in keratinocytes.

Pemphigus is strongly linked to a rare haplotype of HLA-DR4 (DRB1*0402), and this molecule has been shown to present a peptide of desmoglein-3, which other DR4 subtypes cannot. This is therefore a clear example of an autoimmune disease producing pathology by type II mechanisms.

In myasthenia gravis autoantibodies to acetylcholine receptors cause muscle weakness

Myasthenia gravis, a condition in which there is extreme muscular weakness, is associated with antibodies to the acetylcholine receptors on the surface of muscle membranes. The acetylcholine receptors are located at the motor endplate where the neuron contacts the muscle. Transmission of impulses from the nerve to the muscle takes place by the release of acetylcholine from the nerve terminal and its diffusion across the gap to the muscle fiber.

It was noticed that immunization of experimental animals with purified acetylcholine receptors produced a condition of muscular weakness that closely resembled human myasthenia. This suggested a role for antibody to the acetylcholine receptor in the human disease.

Analysis of the lesion in myasthenic muscles indicated that the disease was not due to an inability to synthesize acetylcholine, nor was there any problem in secreting it in response to a nerve impulse – the released acetylcholine was less effective at triggering depolarization of the muscle (Fig. 24.12).

Fig. 24.12 Myasthenia gravis

Normally a nerve impulse passing down a neuron arrives at a motor endplate and causes the release of acetylcholine (ACh). This diffuses across the neuromuscular junction, binds ACh receptors (AChR) on the muscle, and causes ion channels in the muscle membrane to open, which in turn triggers muscular contraction. In myasthenia gravis, antibodies to the receptor block binding of the ACh transmitter. The effect of the released vesicle is therefore reduced, and the muscle can become very weak. Antibody blocking receptors are only one of the factors operating in the disease.

Examination of neuromuscular endplates by immunochemical techniques has demonstrated IgG and the complement proteins, C3 and C9, on the postsynaptic folds of the muscle (Fig. 24.13).

Fig. 24.13 Motor endplate in myasthenia gravis

(1) Electron micrograph showing IgG deposits (G) in discrete patches on the postsynaptic membrane (P). × 13 000. (2) Electron micrograph illustrating C9 (C) shows the postsynaptic region denuded of its nerve terminal – it consists of debris and degenerating folds (D). There is a strong reaction for C9 on this debris. × 9000. (M, muscle fiber.)

(Courtesy of Dr AG Engel.)

Further evidence for a pathogenetic role for IgG in this disease was furnished by the discovery of transient muscle weakness in babies born to mothers with myasthenia gravis. This is significant because it is known that IgG can and does cross the placenta, entering the blood stream of the fetus.

IgG and complement are thought to act in two ways:

Cellular infiltration of myasthenic endplates is rarely seen, so it is assumed that damage does not involve effector cells.

Lambert–Eaton syndrome is a condition with similar symptoms to myasthenia gravis, where the muscular weakness is caused by defective release of acetylcholine from the neuron. In this case the autoantibodies are directed against components of voltage-gated calcium channels or the synaptic vesicle protein synaptogamin. The different forms of Lambert–Eaton syndrome are thought to relate to the target antigen and the class and titer of antibodies involved.