30 Hypersensitivity III contd

Dr. M. N. Gupta

1. Objectives

• Understanding how IgM or IgG mediate hypersensitivity reactions.

•  Explaining how these reactions, also called, cytotoxic hypersensitivity reactions, damage cellular antigens

•  Explaining involvement of opsonization, lysis or antibody-dependent cellular toxicity.
• Understanding immunology behind transfusion reactions.

•  Understanding arthus reaction and serum sickness which represent type III hypersensitivity reactions.

2. Concept map

Vaccinology or Jennerian approach to preventing bacterial infections dominated immunology till the end of 19th century. Around that time, the observations that RBC from another species generated similar immune response as bacteria created sufficient excitement. This was an important finding and broadened the scope of immunological investigations. Hypersensitivity type II reactions include immune responses against erythrocytes and other cells. Unlike type I, type II reactions are not IgE mediated.

As mentioned earlier, the symptoms of type II hypersensitivity appears in 5-8 hrs. time period. Both antibodies and complement participate. Unlike in allergy, in this cytotoxicity IgM or IgG are involved. Once the Ig sensitize the cells, macrophages, N K cells and complement follow depending upon the cells involved.

Blood groups and blood transfusion reactions

The immune response initiated when the erythrocytes from an incompatible donor are injected in the system were the first type II hypersensitivity reactions.

While the seminal work of Landsteiner created ABO blood group system, erythrocytes contain many surface antigens.

These are six major and more common blood group systems. Allotypically variable surface antigens of erythrocytes form the basis of all the systems. Some of these antigens occur on cells of other tissues as well.

Within each system, we have phenotypic variants. In ABO system, the four phenotypic variants are A, B, AB, O. These represent the possible blood types. Individuals having A blood group carry ab against B blood group in the serum. Individuals having B blood group type antibodies against A blood group. Individuals having O blood group have antibodies against both „A‟ and „B‟ antigens.

These antibodies are present even before any blood transfusion. These are believed to be formed as variety of microorganisms happen to carry similar epitopes as „A‟ or „B‟ blood group antigens.

As antibodies to ABO system antigens are present, it necessitates „matching‟ donor blood with the recipient blood before initiating any transfusion reactions.

If the wrong blood is introduced, the specific antibodies react with the donor blood cells and cause agglutination. However, severity of the reaction depends upon the isotype and amount of the antibodies. Prior to transfusion, the antibodies present against the „nonself‟ blood group antigens are usually of IgM type.

Other blood group antigens may also produce IgG. Leukocytes and platelates of the donor blood group also provoke immune response. However responses involving erythrocytes are more severe.

The multiple clinical symptoms of transfusion reactions vary and include fever, nausea and tightness in chest.

IgM immunoglobins cause agglutination, complement activation and intravascular heamolysis.

IgG antibodies may also cause blood cell lysis via complement pathway but it is more likely that they undergo phagocytosis in liver and spleen.

The contents of RBC released in the system can cause acute tubular necrosis of kidney.

In the case of transfusion across the ABO barrier, free Hb can be detected in the plasma within hours. In such cases, transfusion shall be terminated immediately, dieuretic administered to get rid of toxic substances accumulating in the kidney.

What happens when individuals need repeated transfusion of blood even with a matched blood type? These individuals may still develop delayed reactions after 2-6 days of transfusion. These are also type II hypersensitivity reactions. These arise because of the mismatch between donor blood and recipient blood vis a vis other blood group antigens.

The antigen could be Rh, kidd, kell or duffy type. Antibodies are of Ig type and lysis of RBC is by opsonization followed by phagocytosis. The clinical symptoms include increased bilirubin followed by jaundice, fever and anaemia. In such cases no Hb in urine or plasma may be detected as RBC destruction may be limited to extravascular sites.

ABO blood group system antigens are carbohydrate antigens.

The A and B antigen are present as glyco-component of various glycoprotiens on the erythrocyte surface such as anion transport protein and glucose transporter. So, these antigens were not created for matching of blood during transfusion, these were part of other functions!

Hence immune response involved during blood transfusion are unintended consequences as far as biological designs are concerned. These surface antigens are present for some other reasons. This is in line with the overall picture of hypersensitivity unintended consequences or collateral damage as a result of natural biological designs.

Autoimmune haemolytic anaemia

We will cover autoimmune disease separately and extensively. In brief, these diseases represent a pathological situation when immune system forgets the distinction between „self‟ and „nonself‟ antigens. As autoimmune haemolytic anaemia is part of hypersensitivity type II reactions, we will include this in our discussion here.

This type of anaemia is suspected if the patient tests positive in the indirect Coomb‟s test.

The patient produces Ig against its own erythrocytes. The test detects ab bound on the erythrocytes of the patient. The reagent is anti-human Ig which agglutinates the ab coated erythrocytes. Even immune complexes adsorbed on the erythrocytes lead to positive Coomb‟s test.

Autoimmune heamolytic anaemia are divided into three classes on the basis of their causes:

• Warm reactive auto antibodies are reactive only at 37 ºC.
• Cold reactive auto antibodies are reactive only at > 37 ºC.
• Antibodies provoked by drug induced allergic reactions. This is when type I hypersensitivity leads to type II hypersensitivity

Let us understand these classes further.

Warm reactive antibodies

These are generally ab formed against Rh system antigens such as antigenic determinants of RhC, RhE and RhD loci. These ab seems to be directed against different epitopes thant in the case of ab generated during transfusion reactions.

It is beleved that in such cases anaemia results from fast clearance of ab bound RBC by spleen macrophages. Complement mediated lysis is less frequently involved.

Cold reactive antibodies

These are generally present in higher titre than warm reactive ab. Primarily of IgM type ab directed against Ii blood sp system, their action is mediated by complement. Incomplete glycosylation of the core, precursor of A and B antigens, produces I and i epitopes.

The reaction takes place in the peripheral circulation, especially in winter. It causes aggregation and micro thrombosis of small vessels leading to peripheral necrosis in severe case. Anaemia is largely caused by complement mediated lysis in periphery.

How drugs may result in anaemia

Drugs can produce immune response against blood cells including erythrocytes and platelets.

Three routes may be responsible:

• Drugs or its metabolites bind to cell membranes. Ab produced against them will bind followed by complement mediated lysis.

• Immune complex of drugs and ab bind to RBC via immune adherence receptors or Fc region; again complement mediated lysis occurs.

• Drugs bound to cell membranes results in abolition of tolerance and possibly stimulate Th cells. Antibodies against blood group antigens are formed. In the case ab are against the cellular antigens. So once produced, these will destroy even cells to which drug molecules are not bound.

It was Ackroyd who made first observations related to drug induced anaemia in case of the thrombocytopenic perpura. Upon administration of the drug sodormid, purpuric rash because of destruction of platelats was observed.

Wide range of drugs are reported to result in haemolytic anaemia. These include penicillin, quininie and sulphonamides. Luckily incidences are uncommon.

Finally at last example of type II hypersensitivity, let us mention haemolytic disease of newborn (HDN). We have discussed HDN (also called erythroblastosis fetalis) and while discussing Coombs‟ test as a part of tool box of immunology. Let us elaborate on HDN from the perspective of type II hypersensitivity.

Rhesus incompatibility

While HDN can also be caused by incompatibility with other blood group antigens. Those are very rare cases. Generally, RhD (Rhesus D) antigen of erythrocytes is involved.

The problem begins when a Rh- mother has an Rh+ baby and some of the baby‟s blood gets into her circulation. The mother‟s immune system makes ab against Rh antigen. Normally the first baby is not affected. However if pergnanacy with another Rh+ fetus occurs, mothers ab cross over placenta and destroy fetus by attacking its blood cells.

About 85% of the British and US population are Rh+. About 15 % women are Rh- but few factors limit the risk.

The leakage of the Rh+ baby‟s blood through the placenta into the mothers circulation does not occur in adequate amount to initiate immune response. Not all mothers produce anti-Rh antibodies.

If the fathers blood is hetrozygous (if any of his parent was Rh-) w. r. t. Rh antigen, there is only 50% chance that with an Rh- mother, the blood of the fetus will be Rh+ In some cases (around 20%), the formation of antibodies is prevented by interaction of this gene with other blood group genes. A group O Rh- mother will have antibodies against A antigen. If the infants blood was A Rh+, it will be destroyed by mothers antibodies.

Even if the child survives, sever complications arise.

Some measures for preventing HDN are:

Give injections of anti-Rh antibodies to the mother. This kills the erythrocytes of the first baby and except in rare cases ensures that mother does not produce anti Rh0 antibodies.

Factors which promote transplacental haemorrhage include:

• Peeling the placenta of the womb by hand when after birth is delayed

• Attempts to turnaround a poorly placed baby in womb

• Excessive abdominal examination of pregnant women.

• Toxaemia

• Cesarean delivery

• Abortion

Hypersensitivity-type III

This is mediated by immune complexes. All antibody responses of course generate immune complexes. In some cases, these result in type III hypersensitivity. The factors which determine their pathogenic potential are

• Size and amount
• Affinity
• Isotype of ab invoved in forming the complex

During normal course, large complexes formed trigger complement. Mononuclear phagocytic system clears the immune complexes especially in liver when antigen is in excess, the immune complexes do not form extensive network and are small in size. These deposit in blood vessal walls. Two major types of type III reactions are known when imune complexes are deposited locally within a tissue, it is known as Arthus reaction.

Arthus reaction occurs in all tissues but iseasily demonstrated in skin. In a person sensitised to an antigen already has Ig againts it. Some of it diffues out of capilleries. The same antigen injected into skin forms immune complexes which are locally deposited. The mast cells are trigerred via there FcγRIII. Receptors and inflamatory reactions sets in.

Arthus reaction results are visible at the site of injection within few hours of injection. Redness, swelling can be followed by local haemorhage, thrombosis and in severe cases necrosis occurs.

The first step is adherence of neutrophills to vascular endothilium, from there they migrate through the small blood vessels.

In about 6-8 hours, injection site has large number of neutrophills with progressive damage to blood vessels. Escape of fluid causes edema and RBC leakage causes haemorhage.

The immune complexes activate complement which in turn results in platelets releasing vasso active amins. Macrophages release TNF and IL-1. C3a and C5a cause mast cell degranulation and that is how neutrophills are attracted to tissues. Polymorphs release lysozomal enzymes which also contribute to inflamation.

The inflamation is maximal after 5-6 hours and grossly looks as show in figure.

Large immune complexes have Fc moities in the network which bind to mononuclear phagocytic cells with high avidity.

Genetic reasons in some individuals favour production of low affinity anti bodies. Such anti bodies are unable to form large complexes. Carbohydrate components in Ig seem to be important for phagocytosis of corresponding immune complexes. In many diseases like rheumatoid artheritis, abnormality in carbohydrate component affects there clearance. Diseases arising out of deposits of immune complexes can be classified in to three classes.

A low grade persistent infection accompanied by a weak antibody response deposits complexes in the tissue locally. Alpha haemolytic infection by Streptococus viridans , endocarditis due to stephylococal infection and viral hepatitis.

Immunoflouroscence can track formation of these deposits. The renal artery of a patient with chronic hepatitis B was stained with flourescent anti hepatitis antibody and Rhodamine labelled anti IgM.

The second kind of disease is a complication of autoimmune diseases. The prlonged formation of immune complexes overloads the clearance mechanisms and complexes are deposited.

A large number of diseases belonging to each class are known.

In the third kind of diseases, the antigens from moulds, plants and animals are inhaled repeatedly. Farmar‟s lung is an important example of this and occurs due to repeated inhalation of mould from hay. Mouldy hay under damp conditions produces thermophilic actinomycetes like Micropolyspora faeni. Its small pores can penetrate lungs to reach alveoli.

Clinically, Farmar‟s lung is associated with dyspnea and removal of mouldy hay is advised as soon as the disease is detected.

Similarly, “pigeon-breeder‟s lung” arises from continuous inhalation of dust from pigeon faeces. Inhalation of spores from soil actinomycetes during mushroom growing leads to “mushroom –grower‟s disease”. “Librarians lung” can result from inhalation of dust from old books.

Lung section in the case of hypersensitivity pneumonitis due to inhalation od actinomycetes spores show fluid and thicker alveolar walls.

Immune Complex Detection

Histological examination of affected organ is the direct method. Tissue samples can be examined by immunofluorescence.

Circulatory complexes are either in free state or bound to erythrocytes. The later population is on the way to getting cleared, hence looking at the former is of greater clinical importance.

The free complexes estimation by PEG precipitation method starts with rapid separation of plasma.

Just like ammonium sulphate, PEG can also be used tp precipitate proteins by preferential exclusion phenomenon. At 2% PEG concentration, the immune complexes are selectively precipitated. The precipitate is redissolved and Ig quantified by nephelometry or RIA.

Other methods exploits affinity of immune complexes for the complement C1q or Fc receptor on platelets or C3 receptors on BO cell tumour cells.

C1q, for example, can be bound to a polystyrene tube or microplate. The immune complexes when added bind to C1q via their array of Fc. RIA with labelled anti-Ig quantifies the immune complexes.

One drawback is that patient may have free antibodies wich can bind to C1q. We may recall that C1q has structural component similar to collagen. Patients with connective tissue diseases often will have antibodies to collagen.

Finally, clinical evaluation requires careful assessment. Many freely circulating immune complexes are harmless, the damage occurs when some of these start depositing in the tissues!

Serum Sickness

In the era before antibiotics became common, for many diseases, such as diphtheria, a frequent treatment was to inject massive dose of antibody-passive immunization. This serum therapy caused a complication which was called serum sickness. The series of side effects were seen after about 10 days:

Generalized vasculitis with erythema, edema, urticularia of skin, neutropenia, lymph nodee enlargement, joint swelling and proteinuria. These normally subside after few days.

Serum sickness induced in rabbits with a high intravenous dose of antigen (BSA) showed that immune complexes were present in circulation in large doses when serum sickness occurred.

The various effects of immune complexes deposition in various tissues lead to various side effects which have been mentioned.

In transient glomerulonephritis, large complexes under the conditions of excess antigen enetr vascular endothelium resulting in endothelial cell swelling and proliferation. If antigen is in great excess, smaller complexes penetrate even basement membranes. Local damage occurs through complement action.

In arteries, Arthus type lesions develop. It is believed that atransient hypersensitivity type I may be involved.

Repeated small doses of antigen induce chronic serum sickness in rabbits. Macrophages known as mesangial cells have receptors for both complement and immune complexes. These proliferate by IL-1 secreted by themselves upon encountering immune complexes.

Proliferation of mesangial cells and deposits of immune complexes in glomerulis can be seen in these figures.

It must be becoming apparent that while each type of hypersensitivity is initiated at the molecular level differently, the molecules/cells participating in th etissue damage are not very different. Some are prominent in one type, others participate more vigorously in other types. The organs/tissue damaged also may vary.

IN fact, in many diseases more than one type of hypersenstivity may be involved.

Summary

•  IgM and IgG mediatetype II hypersensitivity either directly or through complement. The tissue damage (cytotoxicity) occurs through NK cells, platelets, neutrophils and macrophage.

•  Blood transfusion reactions, autoimmune haemolytic anaemia and RhD problem involve type II hypersensitivity

•  Type III hypersensitivity is mediated by immune complexes deposition in tissues or causing a systemic reaction.

•  Passive immunization causes serum sickness which is also initiated by immune complexes of small or medium size penetrating tissue.