TNFα enhances cell-mediated immune responses in various ways (see Chapter 9). Treatment with antibodies to TNF can reduce severity in the Arthus reaction and, interestingly, anti-TNF therapy has become a highly effective disease modifying agent in treating rheumatoid arthritis.

The ratio of antibody to antigen is directly related to the severity of the ensuing reaction. Complexes formed in either antigen or antibody excess are much less toxic than those formed at equivalence.

Immune complexes clearance by the mononuclear phagocyte system

Immune complexes are opsonized with C3b following complement activation, and removed by the mononuclear phagocyte system, particularly in the liver and spleen. Removal is mediated by the complement C3b receptor, CR1.

In primates, the bulk of CR1 in blood is found on erythrocytes. (Non-primates do not have erythrocyte CR1, and must therefore rely on platelet CR1.) There are about 700 receptors per erythrocyte, and their effectiveness is enhanced by the grouping of receptors in patches, allowing high-avidity binding to the large complexes.

CR1 readily binds immune complexes that have fixed complement, as has been shown by experiments with animals lacking complement (Fig. 25.6).

Fig. 25.6 Effects of complement depletion on handling of immune complexes

A bolus of immune complexes was infused into the circulation of a primate. In animals with a normal complement system the complexes were bound quickly by the CR1 on erythrocytes. In animals whose complement had been depleted by treatment with cobra venom factor, the erythrocytes hardly bound immune complexes at all. Paradoxically, this results in slightly faster removal of complexes in the depleted animals, with the complexes being deposited in the tissues rather than being removed by the spleen.

(Based on data from Waxman FJ, et al. J Clin Invest 1984;74:1329–1340.)

In normal primates the erythrocytes provide a buffer mechanism, binding complexes that have fixed complement and effectively removing them from the plasma. In small blood vessels ‘streamline flow’ allows the erythrocytes to travel in the center of the vessel surrounded by the flowing plasma. Thus it is only the plasma that makes contact with the vessel wall. Only in the sinusoids of the liver and spleen, or at sites of turbulence, do the erythrocytes make contact with the lining of the vessels.

The complexes are transported to the liver and spleen, where they are removed by fixed tissue macrophages (Fig. 25.7). Most of the CR1 is also removed in the process, so in situations of continuous immune complex formation the number of active receptors falls rapidly, impairing the efficiency of immune complex handling.

Fig. 25.7 Clearance of immune complexes in the liver

¹²⁵I-BSA/anti-BSA complexes were infused into a primate over a period of 120 seconds. Blood was sampled from renal, portal, and hepatic veins, and the level of immune complexes bound to the erythrocytes was measured by radioactive counting. The levels of complexes in the renal and portal veins were similar to that in arterial blood. However, complexes were virtually absent from hepatic venous blood throughout, indicating that complexes bound to erythrocytes are removed during a single transit through the liver.

(Based on data from Cornacoff JB, et al. J Clin Invest 1983;71:236–247.)

In patients with SLE, for example, the number of receptors may well be halved. With fewer complement receptors the complexes are cleared rapidly to the liver, but these complexes, which arrive directly rather than on red cells, are later released into the circulation again and may then deposit in the tissues elsewhere and lead to inflammation.

Complexes can also be released from erythrocytes in the circulation by the enzymatic action of factor I.

Q. What action does factor I have in the complement system?

A. It cleaves C3b and C4b into fragments (see Fig. 4.7).

This action leaves a small fragment (C3dg) attached to the CR1 on the cell membrane. These soluble complexes are then removed by phagocytic cells, particularly those in the liver, bearing receptors for IgG Fc (Fig. 25.8).

Fig. 25.8 Immune complex clearance

(1) Immune complex clearance in a healthy normal subject. (2) Immune complex clearance in a patient with SLE. Radiolabeled soluble complexes were injected intravenously and immune complex localization monitored by dynamic imaging. In the normal subject complexes remained longer in the blood through binding to CR1 on red cells, followed by clearance to the liver and the spleen, where immune complexes take part in immunoregulation. In the hypocomplementemic patient with SLE there was little binding to red cells, but rapid clearance to organs such as the liver, with little localizing to the spleen, leading to impaired immunoregulation, which may be a factor in the persistence of autoimmunity.

Complement solubilization of immune complexes

It has been known since Heidelberger’s work on the precipitin curve in the 1930s that complement delays precipitation of immune complexes, though this information was forgotten for a long time.

The ability to keep immune complexes soluble is a function of the classical complement pathway. The complement components reduce the number of antigen epitopes that the antibodies can bind (i.e. they reduce the valency of the antigen) by intercalating into the lattice of the complex, resulting in smaller, soluble complexes. In primates these complement-bearing complexes are readily bound by the C3b receptor (CR1) on erythrocytes.

Complement can rapidly resolubilize precipitated complexes through the alternative pathway. The solubilization appears to occur by the insertion of complement C3b and C3d fragments into the complexes.

It may be that complexes are continually being deposited in normal individuals, but are removed by solubilization. If this is the case, then the process will be inadequate in hypocomplementemic patients and lead to prolonged complex deposition.

Solubilization defects have indeed been observed in sera from patients with systemic immune complex disease, but whether the defect is primary or secondary is not known.

Q. What evidence implies that defective solubilization of complexes is a primary cause of immune complex disease?

A. Genetic deficiency of classical pathway components (i.e. primary defects) are associated with SLE and some other immune complex diseases (see Figs. 4.16, 16.13, and Chapter 20).

Complement deficiency impairs clearance of complexes

In patients with low levels of classical pathway components there is poor binding of immune complexes to erythrocytes. The complement deficiency may result from:

• depletion, caused by immune complex disease; or

• a hereditary disorder, as is the case in C2 deficiency.

This might be expected to result in persistent immune complexes in the circulation, but in fact the reverse occurs, with the complexes disappearing rapidly from the circulation. These non-erythrocyte-bound complexes are taken up rapidly by the liver (but not the spleen) and are then released to be deposited in tissues such as skin, kidney, and muscle, where they can set up inflammatory reactions (Fig. 25.9).

Fig. 25.9 Immune complex transport and removal

In primates, complexes solubilized by complement are bound by CR1 on erythrocytes and transported to the liver where they are removed by hepatic macrophages. Complexes released from erythrocytes by factor I are taken up by cells (including macrophages) bearing receptors for Fc and complement.

Infusion of fresh plasma, containing complement, restores the clearance patterns to normal, illustrating the importance of complement in the clearance of immune complexes.

Failure to localize in the spleen not only results in immune complex disease, but may also have important implications for the development of appropriate immune responses. This is because the spleen plays a vital role in antigen processing and the induction of immune responses (see Chapter 2).

The size of immune complexes affects their deposition

In general, larger immune complexes are rapidly removed by the liver within a few minutes, whereas smaller complexes circulate for longer periods (Fig. 25.10). This is because larger complexes are:

• more effective at binding to Fc receptors and at fixing complement, so binding better to erythrocytes;

• released more slowly from the erythrocytes by the action of factor I.

Fig. 25.10 Complex clearance by mononuclear phagocytes

Large immune complexes are cleared most quickly because they present an IgG–Fc lattice to mononuclear phagocyte cells with Fc receptors, permitting higher avidity binding to these cells. They also fix complement better than small complexes.

Anything that affects the size of complexes is therefore likely to influence clearance.

It has been suggested that a genetic defect that favors the production of low-affinity antibody could lead to the formation of smaller complexes, and so to immune complex disease.

Antibodies to self antigens may have low affinity and recognize only a few epitopes. This results in small complexes and long clearance times because the formation of large, cross-linked lattices is restricted.

Affinity maturation is dependent on efficient somatic mutation and selection of B cells within germinal centers following binding of antigen. This process is far more effective when B cells are stimulated by antigen or immune complexes coated with complement. Patients with complement deficiencies are particularly prone to develop immune complex disease and recent evidence indicates that another way that this is brought about is through poor targeting of antigen complexes to germinal centers, so preventing affinity maturation.

Immunoglobulin classes affect the rate of immune complex clearance

Striking differences have been observed in the clearance of complexes with different immunoglobulin classes:

• IgG complexes are bound by erythrocytes and are gradually removed from the circulation;

• IgA complexes bind poorly to erythrocytes, but disappear rapidly from the circulation, with increased deposition in the kidney, lung, and brain.

Q. Provide an explanation for the different patterns of localization of immune complexes containing IgG and those containing IgA

A. IgG-containing immune complexes activate the complement classical pathway and can bind to CR1 on erythrocytes. IgA does not activate the classical pathway, but can bind to Fcα receptors on mononuclear phagocytes (see Fig. 3.15).

Phagocyte defects allow complexes to persist

Opsonized immune complexes are normally removed by the mononuclear phagocyte system, mainly in the liver and spleen. However, when large amounts of complex are present, the mononuclear phagocyte system may become overloaded, leading to a rise in the level of circulating complex and increased deposition in the glomerulus and elsewhere.

Defective mononuclear phagocytes have been observed in human immune complex disease, but this may be the result of overload rather than a primary defect. In SLE, defects in macrophage clearance of apoptotic debris increase the exposure of intracellular constituents to the immune system. Immune complexes formed between autoantibodies and nucleic acids from apoptotic material can activate plasmacytoid dendritic cells which then produce large quantities of pro-inflammatory Type I interferons – a hallmark cytokine in SLE.

Dendritic cells can also capture immune complexes containing DNA fragments via FcγRIII receptors and TLR 9, generating TNFα production in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF). Dendritic cells can also be activated by immune complexes containing RNA fragments, which activate intracellular TLR7.

Carbohydrate on antibodies affects complex clearance

Carbohydrate groups on immunoglobulin molecules have been shown to be important for the efficient removal of immune complexes by phagocytic cells.

Abnormalities of these carbohydrates occur in immune complex diseases such as rheumatoid arthritis, thus aggravating the disease process. Oligosaccharides associated with the Fc region of IgG lack the normally terminating galactose residue, enhancing rheumatoid factor binding and mannan-binding protein has been shown to bind agalactosyl IgG and subsequently activate complement.

Immune complex deposition in tissues

Immune complexes may persist in the circulation for prolonged periods of time. However, simple persistence is not usually harmful in itself; the problems start only when complexes are deposited in the tissues.

Two questions are relevant to tissue deposition:

• Why are complexes deposited?

• Why do complexes show affinity for particular tissues in different diseases?

The most important trigger for immune complex deposition is probably an increase in vascular permeability

Animal experiments have shown that inert substances such as colloidal carbon will be deposited in vessel walls following the administration of vasoactive substances, such as histamine or serotonin. Circulating immune complexes are deposited in a similar way following the infusion of agents that cause the liberation of mast cell vasoactive amines (including histamine). Pretreatment with antihistamines blocks this effect.

In studies of experimental immune complex disease in rabbits, long-term administration of vasoactive amine antagonists, such as chlorpheniramine and methysergide, has been shown to reduce immune complex deposition considerably (Fig. 25.11). More importantly young NZB/NZW mice which normally develop proteinuria by 9 months old, have less renal pathology, when treated with methysergide. Methysergide blocks the formation of the vasoactive amine 5-hydroxytryptamine (5-HT), and thus blocks a variety of inflammatory events (e.g. deposition of complexes, neutrophil infiltration of capillary walls, and endothelial proliferation), all of which produce the glomerular pathology.

Fig. 25.11 Effect of a vasoactive amine antagonist on immune complex disease

Serum sickness was induced in rabbits with a single injection of bovine serum albumin. The animals were either untreated, platelet depleted, or treated with drugs to block vasoactive amine action. The incidence of serum sickness lesions in the heart and lung was scored. Drug treatment considerably reduced the signs of disease by lowering vascular permeability and thus minimizing immune complex deposition.

Increases in vascular permeability can be initiated by a range of mechanisms, which vary in importance, depending on the diseases and species concerned. This variability makes interpretation of some of the animal models difficult. In general, however, complement, mast cells, basophils, and platelets must all be considered as potential producers of vasoactive amines.

Immune complex deposition is most likely where there is high blood pressure and turbulence

Many macromolecules deposit in the glomerular capillaries, where the blood pressure is approximately four times that of most other capillaries (Fig. 25.12).

Fig. 25.12 Hemodynamic factors affecting complex deposition

Factors that affect complex deposition include filtration and high blood pressure, both of which occur in the formation of ultrafiltrate in the renal glomerulus (1). Turbulence at curves or bifurcations of arteries (2) also favors deposition of immune complexes.

If the glomerular blood pressure of a rabbit is reduced by partially constricting the renal artery or by ligating the ureter, deposition is also reduced. If the glomerular blood pressure is increased by experimentally induced hypertension, immune complex deposition is enhanced as shown by the development of serum sickness. Elsewhere, the most severe lesions also occur at sites of turbulence:

• at turns or bifurcations of arteries,

• in vascular filters such as the choroid plexus and the ciliary body of the eye.

Q. Why should the site of bifurcation in an artery be more susceptible to damage than other sites?

A. This site is subject to high pressure and more erratic shear forces, which may affect the integrity of the endothelium (see Fig. 25.12). In addition blood cells and platelets are not segregated from the vessel wall by laminar flow.

Affinity of antigens for specific tissues can direct complexes to particular sites

Local high blood pressure explains the tendency for deposits to form in certain organs, but does not explain why complexes are deposited on specific organs in certain diseases. In SLE, the kidney is a particular target, whereas in rheumatoid arthritis, although circulating complexes are present, the kidney is usually spared and the joints are the principal target.

It is possible that the antigen in the complex provides the organ specificity, and a convincing model has been established to support this hypothesis. In the model, mice are given endotoxin causing cell damage and release of DNA, which then binds to healthy glomerular basement membrane. Anti-DNA is then produced by polyclonal activation of B cells, and is bound by the fixed DNA leading to local immune complex formation (Fig. 25.13). The production of rheumatoid factor (IgM anti-IgG) allows further immune complex formation to occur in situ.

Fig. 25.13 Tissue binding of antigen with local immune complex formation

Endotoxin injected into mice increases vascular permeability and induces cell damage and release of DNA. The DNA can then become deposited (1) on the collagen of the glomerular basement membrane (GBM) in the kidney. Endotoxin can also induce a polyclonal stimulation of B cells, some of which produce autoantibodies such as anti-DNA and anti-IgG – the latter are known as rheumatoid factors (RFs). Anti-DNA antibody can then bind to the deposited DNA forming a local immune complex (2). RFs have a low affinity for monomeric IgG, but bind with high avidity to the assembled DNA–anti-DNA complex (3). Thus further immune complex formation occurs in situ.

It is possible that in other diseases antigens will be identified with affinity for particular organs.

The charge of the antigen and antibody may be important in some systems. For example, positively charged antigens and antibodies are more likely to be deposited in the negatively charged glomerular basement membrane.

The degree of glycosylation also affects the fate of complexes containing glycoprotein antigens because certain clearance mechanisms are activated by recognition of sugar molecules (e.g. mannan-binding protein).

In certain diseases the antibodies and antigens are both produced within the target organ. The extreme of this is reached in rheumatoid arthritis, where IgG anti-IgG rheumatoid factor is produced by plasma cells within the synovium; these antibodies then combine with each other (self-association), so setting up an inflammatory reaction.

The site of immune complex deposition depends partly on the size of the complex

The fact that the site of immune complex deposition depends partly on the size of the complex is exemplified in the kidney:

• small immune complexes can pass through the glomerular basement membrane, and end up on the epithelial side of the membrane;

• large complexes are unable to cross the membrane and generally accumulate between the endothelium and the basement membrane or the mesangium (Fig. 25.14).

Fig. 25.14 Immune complex deposition in the kidney

The site of complex deposition in the kidney is dependent on the size of the complexes in the circulation. Large complexes become deposited on the glomerular basement membrane, whereas small complexes pass through the basement membrane and are seen on the epithelial side of the glomerulus.

The size of immune complexes depends on the valency of the antigen, and on the titer and affinity of the antibody.

The class of immunoglobulin in an immune complex can influence deposition

There are marked age- and sex-related variations in the class and subclass of anti-DNA antibodies seen in SLE.

Fig. 25.15 Antibody classes in immune complex disease

Immune complex disease is automatic in the NZB/NZW mouse and follows a class switch during early development, from IgM to IgG2a. The graphs show the proportions of anti-DNA antibodies of the IgM and IgG2a isotypes in females and males. Both the class switch and fatal renal disease occur earlier in the female mice of this strain.

Diagnosis of immune complex disease

The ideal place to look for immune complexes is in the affected organ (Figs. 25.2, 25.16).

Tissue samples may be examined by immunofluorescence for the presence of immunoglobulin and complement. The composition, pattern, and particular area of tissue affected all provide useful information on the severity and prognosis of the disease. For example:

• patients with the continuous, granular, subepithelial deposits of IgG found in membranous glomerulonephritis have a poor prognosis with prolonged heavy proteinuria;

• in contrast, those whose complexes are localized in the mesangium have a good prognosis and respond to immunosuppressive therapies.

Not all tissue-bound complexes give rise to an inflammatory response; for example, in SLE complexes are frequently found in skin biopsies from normal-looking skin, as well as from inflamed skin.

Assays for immune complexes in serum are more readily performed than in-situ immunofluorescence, although the results have to be interpreted carefully (see Method box 25.1 ).

Method box 25.1 Assays for circulating immune complexes

Circulating complexes are found in two separate compartments:

• bound to erythrocytes; and

• free in plasma.

Erythrocyte-bound complexes are less likely to be damaging, so it is of more interest to determine the level of free complexes. Care is required when collecting the sample – bound complexes can easily be released during clotting by the action of factor I. To obtain accurate assays of free complexes, the erythrocytes should be rapidly separated from the plasma to prevent the release of bound complexes.

Circulating complexes are often identified by their affinity for complement C1q, using either radiolabeled C1q or solid-phase C1q. Complexes can also be detected by their low solubility in polyethylene glycol (Figs MB25.1.1 and MB25.1.2).

Fig. MB25.1.1 An assay for soluble immune complexes based on polyethylene glycol (PEG)

Polyethylene glycol (PEG) is added to the test serum containing IgG complexes and IgG monomer. When the concentration of PEG reaches 2%, complexes are selectively precipitated; the free antibody remains in solution. The test tube is then centrifuged and the complexes form a pellet at the bottom. The supernatant containing free antibody is removed. The precipitate is washed and redissolved so that the amount of complexed IgG can be measured (e.g. by single radial immunodiffusion, nephelometry, or radioimmunoassay (see Fig. MB25.1.2).

Fig. MB25.1.2 Radioimmunoassay for soluble immune complexes

A three-layer radioimmunoassay for immune complexes based on the use of C1q. (1) C1q is linked to an inert solid phase support, usually a polystyrene tube or plate. (2) Serum containing complexes is added. The complexes bind to the solid-phase C1q by means of the array of Fc regions presented to the C1q. (3) Radiolabeled anti-IgG antibody is added. The amount of radioactivity remaining on the solid phase after washing is measured in a gamma-counter, and is used to calculate the amount of complex bound to the C1q.

Case history

A 53-year-old woman presented with an inflammatory arthritis with a symmetrical pattern affecting her hands, wrists, knees and feet. She developed a pupuric rash on her lower limbs which was worse after exercise and especially on exposure to cold weather. This was followed by sensations of numbness and tingling in both legs below the knees (Fig. 25.w5).

Fig. 25.w5 Cryoglobulin

Cryoglobulin demonstrated after clotting whole blood at 37°C for one hour then cooling the serum for 48 hours at 4°C. The cryoprecipitate dissolved on re-warming the serum to 37°C and was confirmed on gel electrophoresis as being an IgM-anti-IgG rheumatoid factor.

Clinical examination showed a low grade inflammatory arthritis with synovitis affecting the metacarpophalangeal joints, wrists, knees and metatarsophalangeal joints. She had bilateral knee effusions. There was an extensive rash consisting of palpable purpura on her legs and there was a stocking distribution of sensory loss to the level of the knees with absent ankle reflexes (Fig. 25.w6). There was mild bilateral parotid enlargement and dry mouth and dry eyes were noted with a dry Schirmer’s tear test.

Fig. 25-w6 Cutaneous vasculitic purpura in Sjögren’s syndrome

Cutaneous vasculitic purpura in Sjögren’s syndrome.

Investigations showed a normochromic normocytic anemia, elevated erythrocyte sedimentation rate (ESR) of 96 mm/1st hour with a normal C-reactive protein (CRP) of < 5 mg/dL. She was positive for anti-nuclear antibodies (ANA) < 1:1280 with a speckled pattern on immunofluorescence with positive antibodies to Ro (SSA) and La (SSB) but negative anti-DNA antibodies. Rheumatoid factor was strongly positive but anti-citrullinated protein antibodies were negative. Tests for hepatitis B and C were negative. A cryoglobulin was detected by clotting blood at 37°C and cooling the supernatant at 4°C – the precipitate was confirmed as being an IgM-anti-IgG rheumatoid factor. A skin biopsy confirmed a leukocytoclastic vasculitis and electromyography showed a symmetrical sensory neuropathy.

A diagnosis of primary Sjögren’s syndrome was made and she responded well to low dose prednisolone, hydroxychloroquine and azathioprine. Although she continued to have episodes of cutaneous vasculitis, the flares were mild and intermittent.

Critical thinking: Type III serum sickness after factor IX administration (see p. 444 for explanations)

An 8-year-old boy with factor IX deficiency has had repeated episodes of bleeding into his joints and skin, despite requiring administration of factor IX. Ten days after receiving a dose, he developed fever, swelling of multiple joints, and a skin rash. On physical exam, his temperature was 39 °C, he had a diffuse maculopapular skin rash involving his torso and extremities, and both elbows and knees were red, warm, and appeared inflamed. His mother thought the appearance and distribution were very different from the typical appearance after either minor trauma or bleeding into his joints, which he had sustained on multiple previous occasions. His pediatrician ordered the following tests (results shown in Table 25.1) and prescribed a short course of corticosteroids.

1. What immunologic mechanisms are involved in this inflammatory reaction after the boy received the factor IX?

2. Why were corticosteroids prescribed?

3. What is the likelihood that this type of reaction will develop again?

4. What measures would you take to prevent this reaction from occurring again?

Table 25.1

Variable	Result (normal range)
C3 (mg/dL)	38 (85–155)
C4 (mg/dL)	4 (12–45)
anti-nuclear antibody	Negative
hemoglobin (g/dL)	11.2
white cell count (cells/mm³)	11 000
eosinophils (%)	1

He responds to treatment and his symptoms resolve, but 1 year later his mother notices that his face is swollen in the morning and his feet are swollen at the end of the day. Otherwise the boy feels well.

On physical exam, his blood pressure is elevated at 140/90 mmHg and his ankles are very edematous. His joints do not appear inflamed and the skin does not show either evidence of recent bleeding or inflammation. Results of tests are shown in Table 25.2.

5. What immunologic mechanisms are involved in this inflammatory reaction after the boy received the factor IX? How do they differ from the previous episode?

6. What is the likelihood that this type of reaction will develop again?

7. What measures would you take to prevent this reaction again?

Table 25.2

Variable	Result (normal range)
C3 (mg/dL)	142 (85–155)
C4 (mg/dL)	44 (12–45)
anti-nuclear antibody	Negative
hemoglobin (g/dL)	11.6
white cell count (cells/mm³)	8600
eosinophils (%)	< 1
albumin (g/dL)	2.5 (3.5–5.5)
urine protein (g/24 h)	8 (< 0.2)

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Hypersensitivity (Type III)

Immune complex diseases

Persistent infection with a weak antibody response can lead to immune complex disease

Immune complexes can be formed with inhaled antigens

Immune complex disease occurs in autoimmune rheumatic disorders

Cryoglobulins precipitate at low temperature

Immune complexes and inflammation

Q. Aggregation may be directly enhanced by the presence of immune complexes on the basement membrane. How can platelets recognize immune complexes?

Complement is an important mediator of immune complex disease

Autoantibodies to complement components can modulate complement activity

Experimental models of immune complex diseases

Serum sickness can be induced with large injections of foreign antigen

Q. Suggest two reasons why small immune complexes would be cleared more slowly than large complexes

Q. Why would immune complexes deposit in the glomerulus when the complexes are formed outside the kidney?

Autoimmunity causes immune complex disease in the NZB/NZW mouse

The role of Fc receptors in the ‘treatment’ of immune complex disease

Injection of antigen into the skin of pre-sensitized animals produces the Arthus reaction