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12.4E: Type IV (Delayed Cell-Mediated) Reactions - Biology

12.4E: Type IV (Delayed Cell-Mediated) Reactions - Biology


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Type IV hypersensitivity reactions are cell-mediated and take 2 to 3 days to develop.

Learning Objectives

  • Describe Type IV cell-mediated reactions and explain why they take two to three days to develop

Key Points

  • Cell-mediated immunity is an immune response that does not involve antibodies but rather involves the activation of phagocytes, natural killer cells (NK), antigen -specific cytotoxic T-lymphocytes, and the release of various cytokines in response to an antigen.
  • In type IV hypersensitivity reactions, CD4+ helper T cells recognize antigen in a complex with Class 2 major histocompatibility complex on macrophages (the antigen-presenting cells).
  • A classic example of delayed type IV hypersensitivity is the Mantoux tuberculin test in which skin induration indicates exposure to tuberculosis.

Key Terms

  • cellular immunity: Cellular immunity protects the body by: activating antigen-specific cytotoxic T-lymphocytes, activating macrophages and natural killer cells and stimulating cytokine secretion to stimulate other cells involved in adaptive immune responses and innate immune responses.
  • type IV hypersensitivity: A cell-mediated immune response that takes two to three days to develop.

Cell-mediated immunity is an immune response that does not involve antibodies, but rather involves the activation of phagocytes, natural killer cells (NK), antigen-specific cytotoxic T-lymphocytes, and the release of various cytokines in response to an antigen. Historically, the immune system was separated into two branches: humoral immunity, for which the protective function of immunization could be found in the humor (cell-free bodily fluid or serum) and cellular immunity, for which the protective function of immunization was associated with cells. CD4 cells or helper T cells provide protection against different pathogens. Cytotoxic T cells cause death by apoptosis without using cytokines. Therefore in cell mediated immunity cytokines are not always present.

Cellular immunity protects the body by:

1. activating antigen-specific cytotoxic T-lymphocytes that are able to induce apoptosis in body cells displaying epitopes of foreign antigen on their surface, such as virus-infected cells, cells with intracellular bacteria, and cancer cells displaying tumor antigens

2. activating macrophages and natural killer cells, enabling them to destroy pathogens

3. stimulating cells to secrete a variety of cytokines that influence the function of other cells involved in adaptive immune responses and innate immune responses

Cell-mediated immunity is directed primarily at microbes that survive in phagocytes and microbes that infect non-phagocytic cells. It is most effective in removing virus-infected cells, but also participates in defending against fungi, protozoans, cancers, and intracellular bacteria. It also plays a major role in transplant rejection.

Type IV hypersensitivity is often called delayed type hypersensitivity as the reaction takes two to three days to develop. Unlike the other types, it is not antibody mediated but rather is a type of cell-mediated response. CD4+ helper T cells recognize antigen in a complex with Class 2 major histocompatibility complex. The antigen-presenting cells in this case are macrophages that secrete IL-12, which stimulates the proliferation of further CD4+ Th1 cells. CD4+ T cells secrete IL-2 and interferon gamma, further inducing the release of other Th1 cytokines, thus mediating the immune response. Activated CD8+ T cells destroy target cells on contact, whereas activated macrophages produce hydrolytic enzymes and, on presentation with certain intracellular pathogens, transform into multinucleated giant cells.

A classic example of delayed type IV hypersensitivity is the Mantoux tuberculin test in which skin induration indicates exposure to tuberculosis. Other examples include: temporal arteritis, Hashimoto’s thyroiditis, symptoms of leprosy, symptoms of tuberculosis, coeliac disease, graft-versus-host disease and chronic transplant rejection.


Hypersensitivity Types: 4 Important Types of Hypersensitivity

The following points highlight the four important types of hypersensitivity. The types are: 1. Type I Hypersensitivity (Anaphylaxis) 2. Type II Hypersensitivity (Cytotoxic Hypersensitivity) 3. Type III Hypersensitivity 4. Type IV Hypersensitivity.

1. Type I Hypersensitivity (Anaphylaxis):

This type of hypersensitivity is the most common among all the types. About 17% of the human population may be affected, probably due to a natural proneness controlled by the genetic make-up. Anaphylaxis which literally means “opposite of protection” — is mediated by IgE antibodies through interaction with an allergen.

The allergens inciting anaphylaxis include a great variety of substances, like pollens, fibres, insect, venom, fungal spores, house-dust etc. as well as various food materials like egg, milk, fish, crab-meat, peanuts, soybean, various vegetables etc.

Generally, anaphylactic responses are of a mild type producing symptoms, like hay-fever, running nose, skin-eruptions known as “hives” or breathing difficulties. But in some cases, the responses may be severe and may even prove fatal. This latter type of response is called anaphylactic shock.

This may develop within a few minutes (2 to 30 min) and may cause death before any medical help can be provided. Anaphylactic shock is known to result from a bee-sting or intramuscular injection of penicillin. Penicillin itself is not an antigen, but it can act as a hapten.

After combination with serum proteins, it can stimulate allergenic response producing IgE molecules which can combine with the drug. A person sensitized with penicillin may be a victim to anaphylactic shock. The severe form of anaphylaxis is considered as systemic, in contrast to the milder forms which are localized.

During the sensitization phase, the immune system produces B-lymphocytes which are transformed into plasma cells in the usual way. But the plasma cells produce IgE antibodies complementary to the allergenic antigen, instead of normal IgG and IgM antibodies.

The IgE antibodies, so produced, circulate in the blood stream and they become attached to the mast cells and basophils, because IgE antibodies have special affinity for these cells. Mast cells and basophils have a richly granular cytoplasm and each cell has numerous (>100,000) binding sites for IgE molecules.

The IgE antibodies bind to these cells with their Fc domain, while the antigen-binding sites remain free. The sensitization period takes about a week’s time to be completed. During this period millions of IgE molecules are produced and fixed on the mast cells and basophils.

Manifestation of anaphylactic symptoms appears when such a sensitized person is exposed to the same allergen again. The allergen entering into body reacts with the its complementary IgE molecules bound to mast cells and basophils and combine with the antigen binding sites of the antibody.

This interaction causes degranulation of mast cells and basophils and the granules are released in the body fluids. Mast cells occur in close association with the capillaries throughout the body, particularly in the skin and respiratory tracts.

The granules released by mast cells and basophils contain several preformed chemical mediators of which the most important is histamine. The others include heparin, serotonin, bradykinin etc. In addition, some secondary mediators are also produced as a result of the interaction between IgE and an allergen. They include the leucotrienes and prostaglandins.

Degranulation of mast cells and basophils occurs when two IgE molecules are adjacent to each other on these cells and both bind to an antigen (allergen) having the same specificity, thereby forming a bridge. The chemical mediators released by the granules produce various changes associated with allergic response. One of the most important effect is the contraction of smooth muscles.

The small veins are constricted and capillary pores are dilated leading to extrascular accumulation of fluid (edema). The bronchial muscles, as well as those of GI tract, may also contract producing breathing difficulty and cramps. Mast cells present in the mucous membrane of the upper and lower respiratory tracts cause rhinitis and asthma. The events occurring during the sensitization phase and the expression of allergic symptoms after a second encounter with the allergen are diagrammatically shown in Fig. 10.58.

The susceptibility to specific allergens of an individual can be determined by skin-test. It is performed by injecting a small amount of the possible allergens below the skin. A wheal and erythema response indicated by itching, swelling and reddening of the injection spot developed within 2 to 3 minutes and reaching a maximum in about 10 minutes means that the substance is allergenic. Avoidance of the identified allergen(s) is the best way of prevention of anaphylaxis.

Another way of prevention is by desensitization. Once an allergen has been identified, the sensitive person is injected with small doses of the allergen for several weeks. The objective is to build­up immunity to the allergen through production of excess of IgG antibodies, so that they outnumber IgE antibodies. The IgG antibodies in this case are called blocking antibodies, because they block the IgE antibodies to combine with the allergen.

Anaphylaxis can also be prevented by specific drugs. Anaphylactic shock can be prevented by immediate injection of epinephrine. Drugs used for localized anaphylaxis act in two ways. A group of drugs like dexamethasone, prednisolone etc. inhibit the production or release of the chemical mediators responsible for development of allergic symptoms. The other group, mainly the anti-histamines, inhibit the action of chemical mediators, mainly that of histamine.

2. Type II Hypersensitivity (Cytotoxic Hypersensitivity):

This type of hypersensitivity involves IgG antibodies and the complement system and results in cell destruction. IgM may also take part in cell damaging reactions. Cytotoxic hypersensitivity is the result of transfusion of incompatible blood of a donor to a recipient, although this is of rare occurrence because of careful cross-matching of the donor and the recipient’s blood-groups.

A faulty cross­-matching leads to hemolysis of the donor’s erythrocytes in the blood vessels of the recipient. This happens because the alloantigen’s of the donor’s erythrocytes react with the antibodies in the serum of the recipient and in combination with activated complement, the erythrocytes undergo hemolysis.

Similarly, when an Rh-negative recipient is transfused with the blood of an Rh-positive donor, Rh-antibodies develop in the recipient. In case, the same recipient receives subsequently blood from an Rh-positive donor, a rapid and extensive hemolysis occurs in the recipient due to interaction of the Rh-antigen and Rh-antibody. Precaution is necessary that an Rh-negative recipient is not transfused with Rh-positive blood more than once.

Interaction of Rh-antigen and Rh-antibody may lead to a more serious consequence when an Rh-negative mother bears an Rh-positive child, the trait of the child being acquired from an Rh-positive father. Rh-antigen of the fetus enters into mother’s circulation and provokes formation of Rh-antibody in mother.

In a succeeding pregnancy resulting in an Rh-positive fetus, these antibodies enter into the fetal circulation through placenta and react with the Rh-antigen producing serious complications, known as haemolytic disease of the newborn.

The situation leading to this disease is diagrammatically shown in Fig. 10.59:

3. Type III Hypersensitivity:

Normally, the antigen-antibody complex formed as a result of immune reactions is removed by the phagocytic activity of body. However, when bulky antigen-antibody complexes are formed and the aggregates combine with the activated complement, they chemotactically attract the polymorphonuclear leucocytes. These cells release lysosomal enzymes in large quantities to Cause tissue damage. This results in immune complex hypersensitivity (Type III hypersensitivity).

One form of this type of hypersensitivity is the Arthus Reaction. It develops due to deposition of IgG-antigen complexes in the blood vessels causing local damage. When such aggregates are deposited in blood vessels of kidney glomeruli, the result may be nephritis.

Similarly, inhalation of bacteria and fungal spores may give rise to a disease called farmer’s lung. The antigens react with IgG antibodies to form complexes in the epithelial layers of the respiratory tract giving rise to this ailment.

Another form of this type of hypersensitivity is known as lupus (systemic lupus erythematosus). It is produced as a result of interaction of IgG and the nucleoproteins of the disintegrated leucocytes (auto-antigens). Therefore, lupus is an autoimmune disease. The immune-complex may be deposited locally in the skin, or systemically in kidney or heart. Rheumatoid arthritis is another autoimmune disease developing from deposition of immune complexes in the joints.

Serum sickness is another manifestation of immune complex hypersensitivity. Antisera like anti-tetanus serum (ATS) may act as antigen in human body, because these are obtained from animals and are injected to persons for providing immediate protection. The antigen (ATS, for example) can provoke an immune response to produce IgG in the body. These IgG antibodies react with the antisera to produce immune complexes and give rise to serum sickness.

Immune complex hypersensitivity (Type III) is diagrammatically shown in Fig. 10.60:

4. Type IV Hypersensitivity:

In contrast to the first three types of hypersensitivity, Type IV is mediated by cells of immune system, mainly T-cells, but also macrophages and dendritic cells. Furthermore, lymphokines produced by T-cells play an important role. The expression of allergic manifestations takes a longer time, at least 24 hr or more.

Hence, Type IV hypersensitivity is called delayed type of hypersensitivity. The delay in appearance of allergic symptoms after a second exposure to an allergen is mainly due to the time taken by the cellular components to migrate to the site where antigen is present.

The cells involved in delayed hypersensitivity are mainly T-lymphocytes. T-lymphocytes have two main types, — the CD4+ cells and CD8+ cells. The cells involved in Type IV hypersensitivity belong to the CD4+ type. The special group of CD4+ cells taking part in this hypersensitivity are called TD-cells (D standing for delayed hypersensitivity). TD-cells are a part of the T-helper cell (TH-cells) population which constitutes the bulk of CD4+ T-cells. TH-cells are distinguished into TH-1 and TH-2 types, of which TH-2 cells are mainly responsible for activation of B-cells to produce immunoglobulin’s and TH-1 cells are involved in causing the inflammatory responses including delayed hypersensitivity reactions. So, TD-cells belong to the TH-1 type of lymphocytes.

Like the Type I hypersensitivity, Type IV also has two phases: a sensitization phase and an active phase. The allergen can be a microbial antigen or a small molecule that can act as a hapten and can combine with a tissue protein to form an active antigen. The sensitizing antigen binds to some tissue cells and these are ingested by phagocytic cells, like macrophages and dendritic cells. These cells process the antigen and present the antigenic determinants to the TD-cells.

These T-cells recognize the determinants by interacting with the determinants complexed with MHC proteins of the antigen- presenting cells (APC). The close binding between the T-cells and APCs activates the T-cells to proliferate forming a clone including some memory T-cells. Thereby, the person becomes sensitized to the particular allergenic antigen.

In the next phase, the sensitized individual expresses delayed type of hypersensitivity when exposed at din to the same allergen. The memory T-cells activate the sensitized T-cells to produce lymphokines which cause the inflammatory responses associated with Type IV hypersensitivity.

The whole process is diagrammatically shown in Fig. 10.61:

A well-known example of a microbial agent that elicits a delayed hypersensitivity is tuberculin which is a purified protein derivative (PPD) of tubercle bacilli (Mycobacterium tuberculosis). Other microbial agents that stimulate delayed hypersensitivity are Mycobacterium leprae, Brucella and fungi causing histoplasmosis (Histoplasma capsulatum) and candidiasis (Candida albicans).

The tuberculin skin test (Mantoux test) is used to determine if a person has T-cell mediated reactivity towards tubercle bacilli (also known as Koch’s bacilli). In a sensitized individual, an intradermal injection of 0.1 μg of tuberculin results in development of a progressively increasing swollen reddened circular area at the injection site attaining a maximum size in 24 to 72 hr.

Histologically, the response is due to accumulation of large number of inflammatory cells, mainly lymphocytes and macrophages. A positive response shows that the person has immunity to tuberculosis, developed either through active infection or through vaccination and, therefore, does not require BCG vaccination.

Certain low-molecular weight chemical substances can also evoke delayed hypersensitivity. Generally, the allergic symptoms are restricted to the skin and the response is called contact sensitivity. The clinical manifestation is contact dermatitis. A great many varieties of such agents causing contact dermatitis are known. Some examples are metallic nickel and copper, turpentine, formaldehyde, insecticides, detergents, cosmetics, latex, furs, protein fibres etc.

Certain plants, like poison ivy, poison oak etc. can also provoke contact sensitivity. Detection of the possible sensitizing agent can be made by a patch test in which the suspected agent is kept in contact with skin for 24 hr to 48 hr and the skin reaction is examined. Generally, avoidance of the allergenic substance or material removes the adverse effects promptly.


TYPE I HYPERSENSITIVITY

Type I hypersensitivity reaction is commonly called an allergic or immediate hypersensitivity reaction. This reaction is always rapid and can occur within minutes of exposure to an antigen. Type I hypersensitivity reactions are initiated by the interactions between an IgE antibody and a multivalent antigen.

(IgE antibodies are class of antibodies that produces in allergic reactions and multivalent antigen is an antigen molecule with more than one identical epitope per molecule)

Type I hypersensitive reactions can induce by a special type of antigen refer to as allergens which have all the hallmarks of the normal humoral response. Thus, an allergen induces a humoral antibody response, resulting in a generation of antibody secreting plasma cells and memory cells.

Common allergens for type I hypersensitivity are plant pollen, foods (nuts, eggs, seafood, etc.), certain drugs (penicillin, Salicylates, local anaesthetics, dust mites, etc.

Type I reaction can occur in two forms:

The precise component of why some people are more prone to Type 1 hypersensitivity is unclear. However, it has been shown that such individuals preferentially produce more lymphocytes or TH2 cells which in turn favor the change of class to I gE.

1. Anaphylaxis

It is an acute and potentially fatal immediate hypersensitivity reaction. The time of onset of symptoms depends on the level of hypersensitivity and the site of exposure to the antigen. Generally, it affects skin, respiratory tract and cardiovascular system. Plasma cell secretes IgE in response to allergen-specific TH4 cells. This class of antibody binds with high affinity to Fc receptor on the surface of tissue mast cells and basophils. Binding of IgE to the mast cells is also known as sensitization. IgE-coated mast cells can activate on repeat antigen encounter. The primary cellular component in this hypersensitivity are the mast cell, eosinophils, and basophil.

a schematic diagram showing type I hypersensitivity reaction

Further. anaphylaxis has two phases:

Immediate phase

This phase is characterized by degranulation and release of pharmacologically active mediators within minutes of re-exposure to the same antigen. Histamine is the principal biogenic amine that causes rapid vascular and smooth muscle reactions.

Late phase

This phase begins to develop 4–6 hours after the immediate phase reaction and can persist for 1–2 days. It is identified by the infiltration of neutrophils, macrophages, eosinophils, and lymphocytes to the site of reaction.

2. Atopy

Unlike anaphylaxis, atopy is periodic and nonfatal immediate hypersensitivity reaction. Atopic individuals produce high levels of IgE in response to allergens as compared to normal individuals who do not. An example of atopic reactions is bronchial asthma. Atopic hypersensitivity does not transfer through lymphoid cells but it can transfer by serum.

Symptoms

Some of the common symptoms are skin rashes, tingling around the mouth, diarrhoea, etc., It can affect various organs of the body including skin (Urticaria and Eczema), eyes (conjunctivitis) and nose (rhinorrhea.

Generally, it took 10-30 minutes for the symptoms to appear and occasionally it may take up to 10-12 hours.

Diagnosis

Its diagnosis may include skin tests like puncture and intradermal. In addition, measurement of total IgE and IgE antibodies, specific against suspected allergens, also performed.

Treatment

Antihistamines are used for the treatment of type I hypersensitivity. These medications block histamine receptors on cell membrane surfaces.

Treatment for anaphylactic symptoms is injection with epinephrine, a potent neurotransmitter and hormone that effectively halts the immune response.

IgG blockers are also used to treat type I hypersensitivity.


Hypersensitivity is defined as the exaggerated immunological response leading to severe symptoms and even death in a sensitized individual when exposed for the second time. It is commonly termed as allergy. The substances causing allergic/hypersensitivity is known as allergens. Example: Drugs, food stuffs, infectious microorganisms, blood transfusion and contact chemicals.

Classification of Hypersensitivity (Coombs and Gell Classification)

Type I:
Immediate (Atopic or anaphylactic) Hypersensitivity

Type II:
Antibody-dependent Hypersensitivity

Type III:
Immune complex mediated Hypersensitivity

Type IV:
Cell mediated or delayed Hypersensitivity

Type I:
Immediate (Atopic or anaphylactic) Hypersensitivity

This type of hypersensitivity is an allergic reaction provoked by the re-exposure to a specific antigen. The antigen can make its entry through ingestion, inhalation, injection or direct contact. The reaction may involve skin, eyes, nasopharynx and gastrointestinal tract. The reaction is mediated by IgE antibodies (Figure 11.7).

IgE has very high affinity for its receptor on mast cells and basophils. Cross linking of IgE receptor is important in mast cell trigerring. Mast cell degranulation is preceded by increased Ca ++ influx.

Basophils and mast cells release pharmacologically active substances such as histamines and tryptase. This causes inflammatory response. The response is immediate (within seconds to minutes). Hence, it is termed as immediate hypersensitivity. The reaction is either local or systemic.

Allergic rhinitis is commonly known as hay fever. Allergic rhinitis develops when the body’s immune system becomes sensitized and overreacts to something in the environment like pollen grains, strong odour of perfumes, dust etc that typically causes no problem in most people. When a sensitive person inhales an allergen the body’s immune system may react with the symptoms such as sneezing, cough and
puffy swollen eyelids.

Type II Hypersensitivity: Antibody dependent hypersensitivity

In this type of hypersensitivity reactions the antibodies produced by the immune response binds to antigens on the patient’s own cell surfaces. It is also known as cytotoxic hypersensitivity and may affect variety of organs or tissues. Ig G and Ig M antibodies bind to these antigens and form complexes. This inturn activates the classical complement pathway and eliminates the cells presenting the foreign antigen. The reaction takes hours to day (Figure 11.8).

Drug induced haemolytic anaemia Certain drugs such as penicillin, cephalosporin and streptomycin can absorb non-specifically to protein on surface of RBC forming complex similar to hapten-carrier complex. In some patients these complex induce formation of antibodies, which binds to drugs on RBC and induce complement mediated lysis of RBC and thus produce progressive anaemia. This drug induced haemolytic anaemia is an example of Type II hypersensitivity reaction.

Type III Hypersensitivity: Immune complex mediated hypersensitivity

When a huge amount of antigen enters into the body, the body produces higher concentrations of antibodies. These antigens and antibodies combine together to form insoluble complex called immune complex. These complexes are not completely removed by macrophages.

These get attached to minute capillaries of tissues and organs such as kidneys, lung and skin (Figure 11.9). These antigen-antibody complexes activate the classical complement pathway leading to vasodilation. The complement proteins and antigen-antibody complexes attract leucocytes to the area. The leukocytes discharge their killing agents and promote massive inflammation. This can lead to tissue death and haemorrhage.

Arthus reaction

It was first observed by Arthus. It is a local immune complex reaction occurring in the skin. Horse serum and egg albumin are the antigens that induce the arthus reaction. It is characterized by erythema, induration, oedema, haemorrhage and necrosis. This reaction occurs when antibody is found in excess. It appears in 2-8 hours after injection and persists for about 12-24 hours (Table 11.1).

Table 11.1: Difference between Immediate Hypersensitivity and Delayed Hypersensitivity

Immediate Hypersensitivity

Delayed Hypersenstivity

It is often called as delayed hypersensitivity reaction as the reaction takes two to three days to develop. Type IV hypersensitivity is involved in the pathogenesis of many autoimmune and infectious diseases such as tuberculosis and leprosy. T lymphocytes, monocytes and macrophages are involved in the reaction. Cytotoxic T Cells cause direct damage whereas the T helper cells secrete cytokines and activate monocytes and macrophages and cause the bulk damage (Figure 11.10).

Type IV hypersensitivity: Cell Mediated Delayed Hypersensitivity

Tuberculin reaction (Mantoux Reaction)

When a small dose of tuberculin is injected intra dermally in an individual already having tubercle bacilli, the reaction occurs. It is due to the interaction of sensitized T cell and tubercle bacterium. The reaction is manifested on the skin very late only after 48-72 hours.


Immunodeficiency Induced by Plasma Cell Tumors: Comparison of Findings in Human and Murine Hosts*

Susan Zolla-Pazner , in Progress in Myeloma , 1980

Effect on the Secondary Antibody Response

Studies in mice indicate that most PC do not suppress the secondary immune response of their hosts. 14, 40, 47 However, those PC which most severely suppress the primary humoral immune response, also suppress the secondary response. The suppression of the secondary response however is never as severe as the suppression of the primary response. 48

Similar conclusions can be drawn from studies of patients with myeloma. In studies by Cone and Uhr 8 of patients whose pre-immunization sera contained detectable levels of anti-diphtheria toxin, all were able to respond normally to a booster injection of diphtheria toxoid. Similar studies, performed by Fahey et al., 11 showed that 5 out of 7 untreated patients with detectable pre-immunization anti-toxin levels responded to diphtheria toxoid, 3 out of 7 responded to secondary challenge with influenza vaccine and one out of 2 responded to boosting with typhoid vaccine. Thus, the humoral anamnestic response remains unimpaired in many patients, although some patients appear to have a defect in both their primary and secondary antibody responses.


TYPE IV HYPERSENSITIVITY REACTION

Type IV - Hypersensitivity

Type IV hypersensitivity which can also be called delayed-type hypersensitivity (DTH) reaction is a cell-mediated allergic reaction that induces a localized inflammatory reaction through the release of cytokines. In delayed-type hypersensitivity, sensitized T lymphocytes mediate the release of cytokines (e.g., interleukins and interferons) that recruit macrophages to the site of infection or allergen administration. The recruited and activated macrophages release lytic enzymes that cause localized tissue damage at the affected body site as well as contain the activities of the invading allergen. Sensitized T lymphocytes (particularly TDTH cells) are the main effector molecules of the type IV hypersensitivity reaction. Macrophages also act as effector cells of DTH response. Immunoglobulins and complements play no roles in delayed-type hypersensitivity reaction as is obtainable in Type I, II and III hypersensitivity reactions. Unlike other forms of hypersensitivity reactions (inclusive of Type I, II and III reactions as aforementioned) that are immediate-type allergic reactions and occur soon after the injection of the allergen into the host the type IV hypersensitivity reaction occurs hours or days after the allergen or antigen have invaded the animal or human host and thus the name, delayed-type hypersensitivity (DTH) reaction.

There are plethora of pathogenic microorganisms and other substances that induce a delayed-type hypersensitivity reaction. These microbes and substances that causes DTH reaction in humans include: intracellular bacteria (e.g., Mycobacterium tuberculosis, Brucella abortus, Listeria monocytogenes and M. leprae), intracellular viruses (e.g., measles virus, herpes simplex virus and small pox virus or variola), intracellular protozoa (e.g., Leishmania species), intracellular fungi (e.g., Candida albicans, Cryptococcus neoformans, and Histoplasma capsulatum), and other contact allergens such as poison ivy and poison oak that causes several skin reactions in sensitized humans. Delayed-type hypersensitivity (DTH) unlike other forms of hypersensitivity reactions has several protective roles in the animal or human host aside causing localized tissue damage. For example, type IV hypersensitivity defends the host against intracellular microorganisms and other contact allergens.

The recruitment and activation of macrophages in the type IV hypersensitivity reaction to the site of infection is critical in defending the body against intracellular microorganisms which are known to live inside the host cell and away from possible attack by antibodies and other components of the host’s immune system. The intracellular pathogen is eliminated with little damage to the host cell but if the allergen or antigen is not easily removed, an extensive DTH reaction may result in adverse inflammatory reaction that is characteristic of type IV hypersensitivity reaction. Contact dermatitis caused by direct body contact with some plants or flowers (e.g., poison ivy and poison oak tree) is a typical example of delayed-type hypersensitivity reaction. In the hospital, skin testing (e.g., Mantouxtest) based on DTH response is used to determine the sensitivity or sensitization of a person’s immune system to the invasion of an allergen or antigen (in this case: exposure to the tubercle bacilli, Mycobacterium tuberculosis – that causes tuberculosis, TB).

Further reading

William E.P (2003). Fundamental Immunology. 5 th edition. Lippincott Williams and Wilkins Publishers, USA.

Stevens, Christine Dorresteyn (2010). Clinical immunology and serology. Third edition. F.A. Davis Company, Philadelphia.

Silverstein A.M (1999). The history of immunology. In Paul, WE (ed): Fundamental Immunology, 4 th edition. Lippincott Williams and Wilkins, Philadelphia, USA.

Paul W.E (2014). Fundamental Immunology. Seventh edition. Lippincott Williams and Wilkins, USA.

Male D, Brostoff J, Roth D.B and Roitt I (2014). Immunology. Eight edition. Elsevier Saunders, USA.

Levinson W (2010). Review of Medical Microbiology and Immunology. Twelfth edition. The McGraw-Hill Companies, USA.


4 Main Types of Hypersensitivity | Immunology

Several types of hypersensitive reactions can be identified, reflecting differences in the effector molecules generated in the course of the reaction. Gell and Coomb described four types of hyper­sensitivity reactions (Types I, II, III and IV). The first three types are antibody-mediated and the fourth type is mediated mainly by T-cell and macro-phases i.e. cell-mediated (Table 11.1 and 11.2 Fig. 11.2).

1. Type I Hypersensitivity:

Type I hypersensitive reactions are the com­monest type among all types which is mainly induced by certain type of antigens i.e. allergens. Actually anaphylaxis means “opposite of protec­tion” and is mediated by IgE antibodies through interaction with an allergen.

During the activity, this class of antibody (IgE) binds with high affinity to FC (Fragment crystalized) receptors on the surface of constant domains of tissue mast cells and blood basophils. Such IgE-coated mast cells and basophils are said to be sensitized. When the indi­vidual is exposed to the same allergen again, then it cross-links the membrane bound IgE on sensi­tized mast cells and basophils and degranulation of those cells result (Fig. 11.3).

(ii) Biological effects:

1. Normally anaphylactic responses are of a mild type producing symptoms— like hay-fever, running nose, skin erup­tions called as ‘nives’ or breathing diffi­culties.

2. The pharmacologically active mediators released from the granules exert biological effects on the surrounding tissues.

3. In some cases, the responses may be severe, develop within a few minutes (2-30 mins) and may even cause death before any medical help is called anaphylactic shock.

4. The principal effects of vasodilation and smooth muscle contraction may be either systematic or localized.

(iii) Components of type-I reactions:

There are different types of components which are required for type-1 reactions:

3. Mast cells and basophils

4. IgE—binding FC receptors.

5. High—affinity and low-affinity receptors.

(iv) Therapy for Type-I hypersensitivity:

1. The first step in controlling type I is to identify the offending allergen and avoid contact if possible.

2. Removal of house pets, dust-control mea­sures.

3. Repeated injections of increasing doses of allergens called hypo sensitization.

4. Enhancement of phagocytosis by IgG antibody which is referred to a blocking antibody because it competes for the aller­gens, binds and forms a complex that can be removed by phagocytosis.

5. Successful use of anti-histamine drugs result better with respect to type I hyper­sensitivity.

2. Type II Hypersensitivity:

Type II hypersensitive reactions are those in which tissue or cell damage is the direct result of the actions of antibody and complement.

This type of reaction is resulted by blood- transfusion reactions in which host antibodies react with foreign antigens present on the incompatible transfused blood cells and mediate destruction of these cells.

Antibody can mediate cell destruction by activating the complement system to create pores in the membrane of the foreign cell by forming membrane attack complex (MAC). This can also be mediated by antibody dependent cell-mediated cytotoxicity (ADCC).

A faulty cross-matching leads to haemolysis of the donor’s erythrocytes in the blood vessels of the recipient due to the alloantigen of the donor’s erythrocytes react with the antibodies in the serum of the recipient and in combination with activated complement, the erythrocytes undergo haemolysis (Fig. 11.4).

1. Haemolytic disease of the newborn deve­lops when maternal IgG antibodies speci­fic for foetal blood-group antigens cross the placenta and destroy foetal red blood cells. Severe haemolytic disease of the new born is called erythroblastosis foetalis, when an Rh + foetus expresses an Rh antigen on its blood cells that the Rh – mother does not express it (Fig. 11.5).

2. Certain antibiotics (e.g. penicillin, cephalo­sporin and streptomycin) can absorb non- specifically to proteins on RBC mem­branes, forming a complex similar to a hapten-carrier complex and gradually induces anaemia called drug-induced haemolytic anaemia.

3. Type III Hypersensitivity:

When an antigen enters within the body then the antibody reacts with antigen and generates immune complex. This immune complex gradu­ally facilitates removal of antigen by phagocytic activity of body. Large amount of immune com­plexes lead to tissue-damaging Type III hype­rsensitivity. For this reason Type III is called immune complex hypersensitivity.

1. These reactions develop when immune complexes activate the complement sys­tem’s array of immune effector molecules. Complement components (C3a, C4a, C5a) split and produce anaphylatoxins which cause localized mast cell degranulation and increase local vascular permeability.

2. When formed bulky antigen-antibody com­plexes aggregate and combine with the acti­vated complement, they chemotactically attract the polymorphonuclear leucocytes. These cells release lysosomal enzymes in large quantities to cause tissue damage.

1. The recipient of a foreign antiserum deve­lops antibodies, specific for the foreign serum proteins from circulating immune complexes and within days or weeks after exposure to foreign serum antigens, an individual starts to develop serum sick­ness including fever, weakness, vasculitis (rashes) with edema, erythema, lymphadenopathy, arthritis and glomerulo­nephritis.

2. Due to deposition of IgG antigen comple­xes in the blood vessels cause local damage and deposit in blood vessels of kidney glomeruli called Arthus Reaction.

3. Inhalation of bacteria and fungal spores gives rise to a disease called farmer’s lung forming immune complexes in the epithe­lial layers of the respiratory tract.

4. Another type of hypersensitive reaction is known as lupus i.e. systemic lupus erythematosus. It is produced as a result of inter­action of IgG and the nucleoproteins of the disintegrated leucocytes (auto-antigens). Lupus is an autoimmune disease.

4. Type IV Hypersensitivity:

Type IV hypersensitivity is the only type of delayed hypersensitivity. It is mainly controlled by T-cells, macrophages and dendritic cells. It is not the instant response but it is manifested after the second exposure to an allergen. The appea­rance of allergic symptoms come in delay.

Delayed hypersensitivity is maintained by T- lymphocytes. T-cells (lymphocytes) have two main types—the CD4 + cells and CD8 + cells. Type IV hypersensitivity requires CD4 + type. The spe­cial group of CD4 + cells take part in type IV hyper­sensitivity and are called T-D cells (delayed). Again T-helper cell (TH cell) includes T-D cells which constitutes the bulk of CD4 + T-cells. TH cells are again distinguished into TH-1 and TH-2 type, of which TH.2 cells are mainly responsible for acti­vation of B-cell to produce immunoglobulins and TH-1 cells are involved in causing the inflamma­tory responses including delayed hypersensitivity reactions (Fig. 11.6).

1. A microbial agent that elicits a delayed hypersensitivity is tuberculin which is a purified protein derivative (PPD) of tubercle bacilli (Mycobacterium tuberculo­sis). Mycobacterium leprae, the microbial agents also stimulate delayed hyper­sensitivity.

2. The tuberculin skin test (Mantoux test) is used to determine if a person has T-cell mediated reactivity towards tubercle bacilli (also known as Koch’s bacilli).


What is TD Cell Mediated Hyper Sensitivity?

Symptoms of certain hyper sensitivity reactions appear after 24 to 72hours of exposure to allergen and such reactions are referred as delayed type hyper sensitivity reactions.

The term delayed hyper sensitivity was first coined by Koch in late nineteenth century, while studying immunity to tuberculosis. When certain antigens are injected into the skin of sensitized animals an inflammatory response develops after many hours of injection.

Several evidences suggest that, type IV reaction is important in host defense against parasites.

Unlike other forms of hyper sensitivity reactions Type IV hyper sensitivity cannot be transferred from one animal to another thorough serum, but can be transferred by T cells.

Three types of delayed hyper sensitivity reactions have been recognized (1) Contact hyper sensitivity (2) Tuberculin-Type hyper sensitivity (3) Granulomatus hyper sensitivity.

(1) Contact hyper sensitivity:

This kind of hyper sensitivity reaction produces eczematous response at the site of contact with the allergen which is generally a haptane like chemical. The small antigenic molecules (haptanes) penetrate epidermis and bind to the proteins inside and act as sensitizers.

The protein bound haptens are first internalized by epidermal langerhans cells and later carried to paracortical areas of lymphnodes by circulation and finally they are presented to T Cells. Release of lymphokines from the stimulated cells results in manifestation of clinical symptoms after 12 to 15hours of contact.

(2) Tuberculin Type hyper sensitivity (Mantoux Reactions):

When a small dose of tuberculin (antigen derived from the tubercle bacillus) is injected intradermally into a sensitized individual, hardening and swelling develops at the site of injection. Soluble antigens from a number of organisms including Mycobacterium tuberculosis, M leprae and Leishmania tropica induce similar type of reactions in sensitive people. Granulomatous reaction due to persistence of antigens in the tissues may be responsible for the development of lesions.

(3) Granulomatous hyper sensitivity:

It is the most important from of delayed hyper sensitivity reactions rooting many of the pathological effects. If macrophages fail to destroy the engulfed microbes or their Ag particles, the persistent immune complex induces epithelioid cell granuloma formation. In the same way granuloma formation occurs with a variety of foreign bodies or particulate agents, when macrophages fail to digest the inorganic matter of foreign agents or particulate matter. Epithelioid granuloma cells are large flattened cells
with increased endoplasmic reticulum. The region affected by Type IV hyper sensitivity reactions typically show a core of epithelioid cells and macrophages, with or without giant cells and the core is surrounded by a cuff of lymphocytes and a considerable fibrosis. Increased proliferation of fibroblasts and collagen synthesis ensue fibrosis in reaction areas. Delayed hyper sensitivity reaction manifests many chronic diseases in man. Some of the important diseases developed by delayed hyper sensitivity reactions are Tuberculosis, Leprosy, Leishmaniasis, Deep fungal infections and Helminth infections.


Results

Neuroblast reactivation from quiescence, glial growth, and tracheal morphogenesis are nutrient regulated

To better understand how cell growth is coordinated with tissue growth in response to dietary nutrients, we assayed proliferation and growth rates of different cell types in the Drosophila brain in response to animal feeding. Freshly hatched larvae were fed a standard fly food diet for defined periods of time (4, 12, 16, 20, or 24 hours), and growth and proliferation of neuroblasts, glia, and trachea were assayed in the brain (Fig 1). Fly food was supplemented with EdU (5-ethynyl-2′-deoxyuridine) to assay S-phase entry, and size was measured based on the average diameter of neuroblasts or total membrane surface area for glia and trachea (see Materials and Methods for details). After 4 hours of animal feeding, we observed that the 4 mushroom body (MB) neuroblasts (white arrows) and 1 lateral neuroblast were dividing based on their incorporation of EdU and generation of EdU-positive progeny (Fig 1A and 1F and S1A–S1C Fig). In contrast, the other central brain neuroblasts (approximately 100 per brain hemisphere), referred to as non-MB neuroblasts, failed to incorporate EdU during this time (Fig 1A and 1F and S1A–S1C Fig). EdU-positive MB and lateral neuroblasts were larger than the EdU-negative, non-MB neuroblasts, but all expressed the HES1 orthologue, Deadpan (Dpn), a pan-neuroblast marker (S1B and S1C Fig). After 12 hours of feeding, a few non-MB neuroblasts incorporated EdU, and EdU incorporation correlated with increased neuroblast size (Fig 1B and 1F and S1F Fig). Over time, the fraction of EdU-positive, non-MB neuroblasts continued to increase, and at 24 hours, more than 60% of non-MB neuroblasts were EdU-positive (Fig 1C, 1D and 1F). Similar to earlier time points, EdU-positive non-MB neuroblasts were larger than EdU-negative non-MB neuroblasts (S1D and S1F Fig). To confirm that increases in neuroblast size and S-phase entry are nutrient regulated, we fed animals a sucrose-only diet for 24 hours. Consistent with previous reports, no neuroblasts other than the 4 MB neuroblasts (white arrows) and the lateral neuroblast incorporated EdU (Fig 1E and 1F), and non-MB neuroblast size was reduced compared to both EdU-positive and negative non-MB neuroblasts from 24-hour–fed animals (S1E and S1F Fig). We conclude that neuroblasts reactivate from developmental quiescence in response to animal feeding and that reactivation occurs stepwise. Neuroblasts grow in size first and subsequently reenter S-phase and begin generating new progeny. This conclusion is in agreement with previously published work [17,21,22,25,26].

(A–E) Maximum intensity projections of single brain hemispheres. Top panels are colored overlays with single-channel grayscale images below. Brain hemispheres are outlined, and the dotted vertical line indicates the midline. Molecular markers are denoted within panels, and white arrows indicate MB NBs (A,E). (F) Quantification of EdU-positive NBs over time shown as scatter plots with dots representing individual brain hemispheres. Columns indicate mean and error bars indicate SEM in this and all subsequent figures. (G–L) Increases in glial membrane surface area over time in animals expressing mCD8:GFP using repoGAL4. (G–K) Single optical sections of brain hemispheres. Top panels are colored overlays, and bottom panels are single-channel images with mask overlays used in quantification (L) of total glial membrane surface area over time (see Materials and Methods). (M–R) Cerebral tracheal morphology over time in animals expressing GFP using btlGAL4. (M–Q) Maximum intensity projections of brain hemispheres with rendered trachea below and quantified in (R). (F,L,R) One-way ANOVA with Tukey post hoc analysis, *p < 0.05, **p < 0.01, ***p < 0.001. (A,G,M) Scale bar equals 10 μm in this and all subsequent figures. Genotypes of panels listed in S2 Table and data listed in S1 Data. btl, breathless Dpn, Deadpan EdU, 5-ethynyl-2′-deoxyuridine GFP, green fluorescent protein mCD8, membrane-targeted CD8 antigen NB, neuroblast repo, reversed polarity Scrib, Scribble.

Next, we asked whether growth of other cell types within the brain is also nutrient regulated. Nutrient-dependent growth of other cell types, including niche-like cortex glia, could play a role in regulating neuroblast reactivation from quiescence and contribute to the substantial increases in brain size observed after 24 hours of animal feeding (approximately 1.76×, S1G Fig). After 24 hours of feeding, we found that approximately 45% of glia, identified based on expression of the homeodomain transcription factor reversed polarity (Repo), incorporated EdU (S1H and S1I Fig). This was unexpected and suggested that either new glia are being produced or that existing glia endoreplicate in response to feeding. To distinguish between these possibilities, we counted glial nuclei before animal feeding (0 hours fed, freshly hatched) and after 24 hours of animal feeding. We found approximately 95 Repo-positive glia per brain hemisphere before feeding and approximately 100 after feeding, suggesting that EdU incorporation is not followed by glial cell division (S1J Fig). Next, we expressed upstream activating sequence (UAS)–double-parked RNA interference (dupRNAi) in glia (using repoGAL4) to inhibit DNA replication and block endoreplication [27]. After 24 hours of feeding, essentially no dupRNAi-expressing glia were EdU-positive (<1%, n = 5 brain hemispheres), and the glial number was unchanged compared to controls, indicating that glia endoreplicate in response to feeding (S1K and S1L Fig). Next, we expressed a membrane-tagged green fluorescent protein (GFP) in glia (repoGAL4, UAS–membrane-targeted CD8 antigen [mCD8]GFP) to assay glial surface area over time. Between 12 and 24 hours of animal feeding, glial membrane surface area increased nearly 3-fold (Fig 1G–1J and 1L). To determine whether glial membrane growth and S-phase entry are nutrient regulated, animals were fed a sucrose-only diet. After 24 hours, no increases in glia membrane surface area or EdU-positive glia were found, demonstrating that glial membrane growth and S-phase entry are nutrient regulated (Fig 1K and 1L and S1I Fig). We conclude that glia endoreplicate and their membrane surface area increases in response to dietary nutrients.

Next, we assayed growth of brain trachea in response to feeding. Trachea are a network of epithelial-derived tubules that supply oxygen and exchange gas throughout the animal. In the brain, during later larval stages, trachea extend along glia forming a perineuropilar tracheal plexus, analogous to cerebral vasculature in mammals [28]. During mammalian cortical development, intermediate neural progenitors divide near blood vessel branch points, suggesting that cerebral vasculature provides niche-like support, similar to Drosophila glia [29]. To determine whether tracheal morphogenesis in the Drosophila brain is nutrient regulated, we assayed tracheal growth over time in response to feeding. Before animal feeding, a single tracheal branch enters the medial brain region [28]. After 24 hours of feeding, we observed 1 to 4 EdU-positive tracheal nuclei, located at the base of secondary branches, in each brain hemisphere (S1M and S1N Fig), and we found an overall 3-fold increase in tracheal surface area (Fig 1M–1P and 1R). Tracheal branching became more elaborate over time, with brain hemispheres being infiltrated from the inside out in a stereotypic pattern. In contrast, when animals were fed a sucrose-only diet, no EdU-positive tracheal nuclei were observed, and tracheal surface area and branching was reduced (Fig 1Q and 1R and S1N Fig). Together, we conclude that growth (S-phase entry and size) of neuroblasts, glia, and trachea is nutrient regulated. Moreover, growth of all 3 cell types occurs continuously and concomitantly, raising the possibility that nutrient-sensing pathways coordinate growth among different cell types.

Nutrient-dependent growth of neuroblasts, glia, and trachea requires cell-autonomous and non-cell–autonomous activation of PI3-kinase

Increases in cell growth in response to dietary nutrients are typically due to increased PI3-kinase pathway activity, an evolutionarily conserved growth signaling pathway that activates TOR-kinase and other growth pathways [1,3,7,9,14]. To determine whether PI3-kinase is required for nutrient-dependent growth of neuroblasts, glia, and trachea, we expressed UAS-Drosophila protein 60 (dp60) to reduce PI3-kinase activity cell autonomously using cell-type–specific GAL4 lines and assayed EdU incorporation and cell size or membrane surface area after 24 hours of feeding (Fig 2A and S2A–S2F Fig) [30]. When levels of PI3-kinase activity were reduced in neuroblasts (worniu [wor]GAL4, UAS-dp60), neuroblast EdU incorporation was reduced and neuroblast size reduced as reported previously (S2A and S2B Fig) [21,22,26]. When levels of PI3-kinase activity were reduced in all glia (repoGAL4, UAS-dp60), EdU incorporation was essentially absent in glia, and the membrane surface area was reduced (S2C and S2D Fig). Reduction of PI3-kinase activity in trachea (btlGAL4, UAS-dp60) resulted in reduced EdU incorporation and tracheal surface area and branching (S2E and S2F Fig). Therefore, PI3-kinase is required for cell-autonomous nutrient-dependent growth of neuroblasts, glia, and trachea.

(A) Images of segmented brain hemispheres with cell types colored as indicated. (B–D) Single Z images of brain hemispheres. Top panels are colored overlays, and bottom panel are single-channel images overlaid with the mask used for quantification (E) (see Materials and Methods). (F,G,J) EdU-positive NBs after 24 hours of feeding. Maximum intensity projections of brain hemispheres from the indicated genotypes. Top panels are colored overlays, and single-channel grayscale images are below. Brain hemispheres are outlined, and the dotted vertical line to the left indicates the midline. Quantification of EdU-positive NBs (H,K) and NB size (I). (L) Summary of PI3-kinase growth regulation between different cell types in the brain. Circular arrows indicate requirement for autonomous PI3-kinase growth signaling, and straight arrows indicate nonautonomous growth signaling. (E) One-way ANOVA with Tukey post hoc analysis. (H,I,K) Student two-tailed t test, *p < 0.05, **p < 0.01,***p < 0.001, error bars, SEM. Genotypes of panels listed in S2 Table and data listed in S1 Data. btl, breathless Dpn, Deadpan dp60, Drosophila protein 60 EdU, 5-ethynyl-2′-deoxyuridine GFP, green fluorescent protein mCD8, membrane-targeted CD8 antigen NB, neuroblast PI3-kinase, phosphoinositide 3-kinase raptorRNAi, raptor RNA interference repo, reversed polarity UAS, upstream activating sequence wor, worniu.

To determine whether PI3-kinase is also required to coordinate growth among neuroblasts, glia, and trachea in response to nutrition, levels of PI3-kinase activity were reduced in 1 cell type (neuroblasts, glia, or trachea) alone, and the growth of the other 2 cell types was assayed. When PI3-kinase levels were reduced in neuroblasts (worGAL4, UAS-dp60), a modest but significant reduction in glial membrane surface area was observed after 24 hours of feeding (Fig 2A–2C and 2E). To further support that neuroblast growth is required for glial growth, we expressed raptorRNAi in neuroblasts (worGAL4, UAS-raptorRNAi) to knock down TOR activity. Again, a significant reduction in glial membrane surface area was found after 24 hours of feeding (Fig 2D and 2E). Reductions in glial membrane surface area could be due to reductions in glia number because some worGAL4-expressing neuroblast lineages generate glia. Indeed, glia number was reduced compared with controls in raptorRNAi knockdown animals (worGAL4, UAS-raptorRNAi) but remained unchanged when PI3-kinase activity was reduced in neuroblasts (worGAL4, UAS-dp60) (S2G Fig). Next, we assayed tracheal surface area when levels of PI3-kinase activity were reduced in neuroblasts. No change in tracheal surface area was detected (S2H–S2J Fig). We conclude that activation of PI3-kinase signaling in neuroblasts is required for glial membrane growth, but not for growth of trachea.

Next, we reduced PI3-kinase activity in glia and assayed the growth of neuroblasts and trachea. When PI3-kinase levels were reduced in glia (repoGAL4, UAS-dp60), significant reductions in both neuroblast EdU incorporation and neuroblast size were found after 24 hours of feeding (Fig 2F–2I). Yet, tracheal surface area remained relatively unchanged compared to controls (S2K and S2L Fig). Finally, we reduced PI3-kinase activity in trachea and assayed the growth of neuroblasts and glia. When PI3-kinase activity levels were reduced in trachea (btlGAL4, UAS-dp60), we found a modest but significant reduction in neuroblast EdU incorporation after 24 hours of feeding (Fig 2J and 2K) but no change in glia membrane surface area (S2M and S2N Fig). We conclude that activation of PI3-kinase signaling in glia and trachea both contribute to neuroblast reactivation, revealing that both cell types provide niche-like stem cell support. In addition, activation of PI3-kinase in neuroblasts is required for glial growth, but not tracheal growth (summary panel Fig 2L). Altogether, we conclude that PI3-kinase signaling functions in a cell-autonomous and nonautonomous manner to coordinate growth among different cell types within the developing Drosophila brain.

Growth of cortex, subperineurial, and neuropil glia is nutrient regulated and PI3-kinase dependent

The glial population in the Drosophila brain is composed of several different subtypes, including cortex glia that ensheathe neuroblasts and their newborn neuron progeny, SPG that encapsulate the CNS and form part of the "blood-brain–like barrier", and neuropil glia that separate neuron cell bodies from their axon projections (S3A Fig). To better understand how PI3-kinase–dependent growth is coordinated between neuroblasts and glia, we assessed numbers and types of glia, based on location, before and after animal feeding. Before feeding, approximately 95 Repo-positive glia were found in each brain hemisphere. Of these, approximately 18 were cortex glia (19%), approximately 34 were neuropil glia (35%, which include ensheathing glia of the neuropil and astrocytes), and the rest, approximately 46%, were SPG and optic-lobe–associated glia combined. After 24 hours of feeding, glial number and subtype distribution based on location remained relatively similar (S3B Fig). We conclude that glial number and type are not specified by dietary nutrient uptake.

Next, we screened existing glial GAL4 lines to identify lines that drive GAL4 reporter expression specifically in different glial subsets in 24-hour–fed animals (S1 Table). We found that NP0577GAL4 drove UAS-histone red fluorescent protein (RFP) reporter expression in all cortex glia, identified based on location and Repo coexpression, as well as in some other glia (S3C and S3D Fig and S1 Table). MoodyGAL4 drove UAS-histoneRFP in most SPG, almost half of neuropil glia, but no other glia (S3E and S3F Fig and S1 Table). Of the 6 glial GAL4 lines screened, NP0577GAL4 and moodyGAL4 exhibited the most restricted and specific patterns of glia GAL4 expression in brains of 24-hour–fed animals. Therefore, we used these lines for subsequent analyses.

We expressed a membrane-tagged GFP in cortex glia (NP0577GAL4, UAS-mCD8GFP) to assay membrane growth in this glial subtype. After 24 hours of feeding, cortex glial membrane surface area increased nearly 3-fold, and cortex glia began to ensheathe neuroblasts with new glial membrane (Fig 3A and 3D). In contrast, animals fed a sucrose-only diet or a normal diet with reduced PI3-kinase levels in cortex glia (NP0577GAL4, UAS-dp60) showed no increases in cortex glia membrane surface area after 24 hours (Fig 3B–3D). Furthermore, we found no evidence of glial membrane ensheathment of neuroblasts under these conditions (Fig 3B and 3C, right panels). Next, we carried out a similar set of experiments in moodyGAL4, UASmCD8GFP animals. In animals fed a sucrose-only diet or those fed a normal diet but with reduced PI3-kinase levels in glia (moodyGAL4, UAS-dp60), we observed reductions in SPG and neuropil glial membrane surface area compared to animals fed a normal diet for 24 hours (Fig 3E–3H). Of note, neither SPG nor neuropil glia ensheathed neuroblasts (Fig 3E, right panel). We conclude that cortex glia, SPG, and neuropil glia require dietary nutrients and PI3-kinase activity for growth. Importantly, glial-subtype–specific GAL4 lines provide the tools necessary to further dissect nonautonomous growth regulation between neuroblasts and specific glia subtypes in response to nutrition and PI3-kinase activity levels.

(A-C) Cortex glial membrane morphology after 24 hours of feeding standard food (A and C) or sucrose (B). (A–C) Single Z planes of brain hemispheres. Top panels are colored overlays, and bottom panels are single-channel images with the mask overlays used for quantification of cortex glia membrane surface area (D). Boxed Dpn-positive NBs are shown at higher magnification to the right. (E–G) SPG and neuropil glial membrane morphology after 24 hours of feeding on standard food (E and G) or sucrose (F). (J–L) Single Z images of brain hemispheres. Top panels are colored overlays, and bottom panels show single-channel images with the mask overlay used for quantification of SPG and neuropil membrane surface area (H). Dpn-positive NBs denoted in the box shown at higher magnification to the right. One-way ANOVA with Tukey post hoc analysis, **p < 0.01, ***p < 0.001. Error bars, SEM. Genotypes of panels listed in S2 Table and data listed in S1 Data. Dpn, Deadpan dp60, Drosophila protein 60 GFP, green fluorescent protein mCD8, membrane-targeted CD8 antigen NB, neuroblast PI3-kinase, phosphoinositide 3-kinase Scrib, Scribble SPG, subperineurial glia UAS, upstream activating sequence.

Cortex glia are required to reactivate neuroblasts from developmental quiescence

We found that reduction of PI3-kinase activity in all glia inhibited neuroblast reactivation from developmental quiescence (refer back to Fig 2F–2I). Using glial-subtype–specific GAL4 lines, we reduced PI3-kinase levels in a glial-subtype–specific manner and assayed neuroblast EdU incorporation and size after 24 hours of feeding. When PI3-kinase activity was reduced in cortex glia (NP0577GAL4, UAS-dp60), we found that neuroblast EdU incorporation and size were reduced after 24 hours of animal feeding (Fig 4A, 4B, 4D and 4E). The same effect was observed when phosphatidylinositol (3,4,5)-trisphosphate (PIP3) levels were reduced by overexpressing the lipid phosphatase, phosphatase and tensin homolog (Pten) (NP0577GAL4, UAS-Pten) (Fig 4D). However, when PI3-kinase activity levels were reduced in SPG/neuropil glia (moodyGAL4, UAS-dp60), no difference in neuroblast EdU incorporation was found compared to controls (Fig 4F–4H). Next, UAS-dp60 was simultaneously expressed in both cortex glia and trachea. A significant reduction in neuroblast EdU incorporation was found compared to animals expressing UAS-dp60 alone in either cortex glia (NP0577GAL4) or trachea (btlGAL4) (Fig 4I). Finally, to confirm that cortex glia are indeed required for neuroblast reactivation, the proapoptotic gene grim was expressed (NP0577GAL4, UAS-grim) to ablate cortex glia genetically (Fig 4J–4L). After 24 hours of feeding, neuroblast EdU incorporation was essentially absent, and neuroblast size was reduced compared with controls (Fig 4C–4E). We conclude that neuroblast growth and reactivation from quiescence requires activation of PI3-kinase in cortex glia, the glial subtype that ensheathes neuroblasts and their newborn progeny, but not SPG or neuropil glia. Furthermore, activation of PI3-kinase growth signaling in both trachea and cortex glia together promote neuroblast reactivation from developmental quiescence.

(A–D) EdU-positive NBs in animals with genetic manipulations in cortex glia at 24 hours after feeding. (A–C) Maximum intensity projections of brain hemispheres. Top panels are colored overlays, and bottom panels show single-channel grayscale images. Brain hemispheres are outlined, and the dotted vertical lines indicate the midline. Graphs show quantification of EdU-positive NBs per brain hemisphere (D) and NB size based on EdU incorporation (E). Numbers in E indicate the number of NBs analyzed. (F–H) EdU-positive NBs in animals with genetic manipulations in SPG and neuropil glia at 24 hours after feeding. (F–G) Maximum intensity projections of brain hemispheres. Panels are colored overlays (top), with single-channel grayscale images below. Brain hemispheres are outlined, and dotted vertical lines indicate the midline. Quantification of EdU-positive NBs per brain hemisphere (H). (I) Quantification of EdU-positive NBs per brain hemisphere of indicated genotypes. (J) Single Z-plane image, top panel is a colored overlay with single-channel grayscale image below, brain hemisphere outlined. (K) Two (top and bottom) grayscale images of same Z-plane, with quantification of cortex glia number following genetic ablation quantified in (L). (D,I) One-way ANOVA with Tukey post hoc analysis and (E,H,L) Student two-tailed t test, *p < 0.05, **p < 0.01, ***p < 0.001. Genotypes of panels listed in S2 Table and data listed in S1 Data. btl, breathless Dpn, Deadpan dp60, Drosophila protein 60 EdU, 5-ethynyl-2′-deoxyuridine mCD8, membrane-targeted CD8 antigen NB, neuroblast O/E, overexpression Pten, phosphatase and tensin homolog Repo, reversed polarity RFP, red fluorescent protein Scrib, Scribble SPG, subperineurial glia UAS, upstream activating sequence.

Dilp-2 regulates neuroblast reactivation and cortex glia membrane growth, but not growth of trachea

PI3-kinase is activated in response to feeding, after any one of the 7 Drosophila insulin-like peptides (Dilps1–7) bind to and activate the single insulin-like tyrosine kinase receptor (InR). While a role in neuroblast reactivation has been attributed to Dilps [21–23], a systematic analysis of each of the dilp mutants is still lacking. To better understand how PI3-kinase coordinates growth among different cell types within the developing brain, we assayed neuroblast EdU incorporation in each of the 7 dilp null mutants. Compared to controls and other dilp mutants, neuroblast EdU incorporation in the brain was reduced in dilp1, dilp2, and dilp7 null mutants after 24 hours of feeding (Fig 5A). Of these, dilp2 mutants displayed the most severe and penetrant reductions in neuroblast EdU incorporation and neuroblast size (Fig 5A–5D and S4A Fig). To confirm that reduced neuroblast EdU incorporation was due to the absence of dilp2 and not a second background mutation, we assayed EdU incorporation in animals transheterozygous for the dilp2 null allele over a small deficiency that removes dilp2 and the neighboring dilp3 locus. The number of EdU-positive neuroblasts after 24 hours of feeding in transheterozygous larvae was indistinguishable from dilp2 1 homozygotes (Fig 5A). We conclude that Dilp-1, Dilp-2, and Dilp-7 regulate neuroblast reactivation from quiescence in the brain.

(A–D) EdU-positive NBs after 24 hours of feeding in dilp single-null–mutant animals with quantification (A) shown as a scatter plot with dots indicating brain hemispheres, bars indicating means, and error bars SEM. (B–D) Maximum intensity projections of brain hemispheres. Top panels are colored overlays with single-channel grayscale images below. Brain hemispheres are outlined, and dotted vertical lines indicate the midline. (E–G) Cortex glial membrane morphology after 24 hours of feeding in controls and dilp2 mutants. (E,F) Single Z images of brain hemispheres. Top panels are colored overlays, and bottom panels are single-channel images with the mask overlays used for quantification in (G). (H–J) Cerebral tracheal morphology after 24 hours of feeding in controls and dilp2 mutants. (H,I) Maximum intensity projections of brain hemispheres with rendered trachea below and quantified in (J). (A,G) One-way ANOVA with Tukey post hoc analysis and (J) Student two-tailed t test, *p < 0.05, **p < 0.01, ***p < 0.001. Genotypes of panels listed in S2 Table and data listed in S1 Data. Dilp, Drosophila insulin-like peptide Dlg, discs-large Dpn, Deadpan EdU, 5-ethynyl-2′-deoxyuridine GFP, green fluorescent protein mCD8, membrane-targeted mCD8 antigen NB, neuroblast n.s., not significant Scrib, Scribble.

Given the prominent role of Dilp-2 in neuroblast growth and reactivation in the brain, we next asked whether Dilp-2 is also required for cortex glial and tracheal growth in response to animal feeding. Compared to controls, cortex glial membrane surface area was reduced by half in dilp2 mutants and in animals transheterozygous for the dilp2 null allele over the dilp2, dilp3 deficiency after 24 hours of feeding (Fig 5E–5G). In contrast, tracheal surface area remained unchanged (Fig 5H–5J). Next, we assayed neuroblast reactivation and cortex glial membrane surface area at 48 hours ALH (after larval hatching). No difference in number of EdU-positive neuroblasts or glial cortex membrane surface was observed between controls and dilp2 mutants (S4B and S4C Fig). Because dilp2 mutants reportedly delay development anywhere from 8 to 16 hours from egg to adult [45], we assayed mouth hook morphology as a proxy for larval stage. No difference between controls and dilp2 mutants was observed at 24 or 48 hours ALH, suggesting that dilp2 null mutants are not developmentally delayed during early larval stages (S4D and S4E Fig). We conclude that Dilp-2 is required for cortex glial membrane growth, but not tracheal growth. Furthermore, because Dilp-2 is also required for neuroblast growth and reactivation, it suggests that growth coordination between cortex glia and neuroblasts is Dilp-2 dependent.

Dilp-2 regulates CNS Dilp-6 protein levels, but not dilp6 transcript levels

Somewhat surprisingly, neuroblast EdU incorporation in brains of dilp6 null-mutant animals was not different compared with controls, dilp3, dilp4, or dip5 mutants, nor was neuroblast size affected by loss of dilp6 (Fig 5A, 5B and 5D and S4A Fig). This was unexpected because Dilp-6 is reported to be synthesized and secreted from SPG in response to animal feeding, leading to PI3-kinase activation and reactivation of adjacent quiescent neuroblasts in the ventral nerve cord (VNC) [21–23]. However, neuroblasts located within different CNS regions, brain versus VNC, could have different requirements for reactivation based on neuroblast intrinsic differences and/or differences in tissue architecture. To determine whether Dilp-6 regulates growth of other cell types within the brain, we assayed glial and tracheal surface area in dilp6 mutants after 24 hours of feeding. No differences were observed compared to controls (Fig 6A–6F). To determine whether Dilp-2 masks Dilp-6 function in the brain, we assayed neuroblast EdU incorporation and size in dilp2, dilp6 double-mutant animals. After 24 hours of feeding, neuroblast EdU incorporation was reduced in dilp2, dilp6 double mutants (approximately 15% EdU-positive neuroblasts) compared with dilp2 single mutants (approximately 22% EdU-positive neuroblasts) however, this reduction was not statistically different (S5A and S5B Fig). Next, we assayed dilp2 and dilp6 transcript levels (Fig 6G and 6H), and endogenous Dilp-2 and Dilp-6 protein levels (Fig 6I) in the CNS of wild-type animals after feeding. We found that Dilp-2 transcript and protein levels were approximately 80 times higher than Dilp-6 transcript and protein levels, consistent with the notion that Dilp-2 is the predominant Dilp in the central brain (Fig 6G–6I). Furthermore, in dilp2 mutants, we found that dilp6 transcript levels were not different from wild-type control levels, although Dilp-6 protein levels were significantly reduced compared to control animals (Fig 6H and 6J). We conclude that Dilp-2 is the primary Dilp regulating PI3-kinase–dependent growth of neuroblasts, glia, and trachea in the brain and that Dilp-2 directly or indirectly regulates Dilp-6 protein levels.

(A–C) Glial membrane morphology after 24 hours of feeding in controls and dilp6 mutants. (A,B) Single Z images of brain hemispheres. Panels are colored overlays (top) and single-channel images with mask overlays (bottom) used for quantification of total glial membrane surface (C). (D–F) Cerebral tracheal morphology after 24 hours of feeding in controls and dilp6 mutants. (D,E) Maximum intensity projections of brain hemispheres with rendered trachea below and quantified in (F). (G,H) RT-qPCR analysis of dilp2 and dilp6 transcript levels in the CNS of wild-type and dilp2 mutant animals after 24 hours of feeding. Transcript levels of dilp2 and dilp6 are normalized to Gapdh1 and then to dilp6 levels in wild-type animals. (I,J) Brain Dilp-2 and Dilp-6 protein levels, normalized to total brain protein, in wild-type and dilp2 mutant animals after 72 hours of feeding. (K) Quantification of EdU-positive NBs in the indicated genotypes after 24 hours of feeding. (C,F,J) Student two-tailed t test (K) one-way ANOVA with Tukey post hoc analysis and **p < 0.01, ***p < 0.001. Genotypes of panels listed in S2 Table and data listed in S1 Data. a.u., arbitrary unit CNS, central nervous system Dilp, Drosophila insulin-like peptide Dpn, Deadpan EdU, 5-ethynyl-2′-deoxyuridine Gapdh1, Glyceraldehyde-3-phosphate dehydrogenase GFP, green fluorescent protein HF, HA-FLAG tag mCD8, membrane-targeted CD8 antigen NB, neuroblast RT-qPCR, real-time qPCR Scrib, Scribble UAS, upstream activating sequence.

Next, we carried out a series of rescue experiments in dilp2 mutants to identify the cell source of Dilp-2 required for neuroblast reactivation. When UAS-dilp2 was expressed in insulin-producing cells (IPCs, dilp2 GAL4), neuroblast EdU incorporation was rescued in dilp2 mutants (Fig 6K). Similarly, expression of UAS-dilp2 in SPG (moodyGAL4) also rescued neuroblast EdU incorporation in dilp2 mutants. Because Dilp-2 is normally expressed in the IPCs [42,43] and not BBB (blood–brain barrier) glia, this suggests that Dilp-2 is required in IPCs for neuroblast reactivation in the brain. In contrast, expression of UAS-dilp6 in IPCs did not rescue neuroblast EdU incorporation in dilp2 mutants. We conclude that Dilp-2 is required in the IPCs for neuroblast reactivation from developmental quiescence in the brain hemispheres.

Coordination of growth between neuroblasts and cortex glia promotes formation of a selective membrane barrier for niche stem cell support

During later stages of larval development, neuroblasts and their progeny reside within characteristic glial membrane-bound pockets (Fig 7A and 7B). One or two cortex glia that lie nearby or adjacent to neuroblasts and their progeny provide the membrane that constitutes each pocket. We injected a fluorescently conjugated 10-kDa dextran directly into the brain to test the permeability of glia membrane-bound pockets. We found fluorescent dextran colocalized with cortex glia membrane along the outside of each pocket, but we did not find fluorescence within the pocket (Fig 7A). This suggests that cortex glia form a membrane barrier that selectively regulates passage of factors based on size.

(A) Top panel, colored overlay with RFP marking glial membranes and GFP marking pcnaGFP-expressing NBs. Middle panel, single-channel grayscale image of 10-kDa dextran. Bottom panel, colored overlay with 10-kDa dextran and RFP marking glial membranes. To the right, high-magnification images of the boxed NB, with double-labeled glial membrane in a 72-hour–fed animal after dextran injection (see Materials and Methods). (B) Cortex glia ensheathe NBs (white bracket) and their newborn progeny, which express high levels of Scrib. Top panel, colored overlay. Middle panel, single-channel image with GFP marking glial membranes. Bottom panel is a depiction of the glial and NB segments used for quantification in (C,D,K) (see Materials and Methods). (C,D) Percentage of NB membrane in contact with glial contact over time. Colored circles in (C) indicate individual NBs, and colored circles in (D) indicate averages for the indicated time points. Both are plotted relative to NB area. (E–G) Increasing surface contact between NBs and glia membrane over time. Single Z-plane of representative NBs at different time points after feeding. Top panels are colored overlays, middles panels are single-channel images, and bottom panels depict the glial NB segment and NB segment used in quantification (C–D). (H–K) Top panel colored overlay, middle panel single-channel image, and bottom panel glial NB segment and NB segment used in quantification of animals at 24 hours of feeding with genotypes listed above. (D,K) One-way ANOVA with Tukey post hoc analysis, ***p < 0.001. Genotypes of panels listed in S2 Table and data listed in S1 Data. Dilp, Drosophila insulin-like peptide GFP, green fluorescent protein mCD8, membrane-targeted CD8 antigen NB, neuroblast pcna, proliferating cell nuclear antigen repo, reversed polarity RFP, red fluorescent protein Scrib, Scribble UAS, upstream activating sequence.

Next, we investigated whether Dilp-2–mediated PI3-kinase activation in neuroblasts and glia is required for glia pocket formation. First, we measured the fraction of neuroblast membrane, marked by endogenous expression of Scribble (Scrib), in contact with glia membrane, marked by transgenic expression of UAS-mCD8:GFP (% neuroblast membrane with glial contact see Materials and Methods) over time in control animals (Fig 7B, schematic at bottom). From 0 hour freshly hatched stages until 8 hours after feeding, we found that the fraction of neuroblast membrane in contact with glial membrane increased approximately 3.5-fold (Fig 7C–7F). At 24 hours after feeding, glia ensheathe approximately 44% of the neuroblast membrane, and at 72 hours after feeding, they ensheathe approximately 72% (Fig 7B–7D and 7G). Next, we assessed the temporal relationship between neuroblast–glial membrane contact and changes in neuroblast size. We found that neuroblast size began to increase 16 hours after feeding, after the fraction of neuroblast–glial contact increased (Fig 7C–7G). Next, we measured the fraction of neuroblast membrane in contact with glia membrane in 24-hour–fed dilp2 mutants (Fig 7I and 7K). We found significant reductions in neuroblast membrane and glia membrane contact that correlated with reductions in neuroblast size. A similar result was found when levels of PI3-kinase activity were reduced in glia, but not in neuroblasts (Fig 7H, 7J and 7K). In neuroblasts, when PI3-kinase activity levels were reduced, neuroblast size remained reduced compared to controls, but increases in neuroblast membrane in contact with glial membrane were found. We conclude that Dilp-2 mediated PI3-kinase activation is required to initiate glial pocket formation. Importantly, increases in neuroblast membrane with glia contact precede increases in neuroblast size, consistent with the notion that cortex glia membrane contact triggers neuroblast growth and reactivation.


12.4E: Type IV (Delayed Cell-Mediated) Reactions - Biology

Type four hypersensitivity reaction is a cell-mediated reaction that can occur in response to contact with certain allergens resulting in what is called contact dermatitis or in response to some diagnostic procedures as in the tuberculin skin test. Certain allergens must be avoided to treat this condition. This activity reviews the evaluation and management of type four hypersensitivity reactions and highlights the role of the interprofessional team in improving care for patients with this condition.

  • Describe the epidemiology of type four hypersensitivity reactions.
  • Summarize the pathophysiology of type four hypersensitivity reactions.
  • Explain the common physical exam findings associated with type four hypersensitivity reactions.
  • Review the importance of collaboration and care coordination amongst the interprofessional team to enhance the care of patients with type four hypersensitivity reactions.

Introduction

Our immune system plays a crucial role in protecting our body against pathogens, but sometimes there is an exaggerated response. This exaggerated response is triggered by the interaction of the immune system with an antigen (allergen) and is referred to as hypersensitivity. Hypersensitivity reactions are classified into four types by Coombs and Gell. The first three types are considered immediate hypersensitivity reactions because they occur within 24 hours. The fourth type is considered a delayed hypersensitivity reaction because it usually occurs more than 12 hours after exposure to the allergen, with a maximal reaction time between 48 and 72 hours. The four types of hypersensitivity are: 

  • Type I: reaction mediated by IgE antibodies
  • Type II: cytotoxic reaction mediated by IgG or IgM antibodies
  • Type III: reaction mediated by immune complexes
  • Type IV: delayed reaction mediated by cellular response 

A Type IV hypersensitivity reaction is mediated by T cells that provoke an inflammatory reaction against exogenous or endogenous antigens. In certain situations, other cells, such as monocytes, eosinophils, and neutrophils, can be involved. After antigen exposure, an initial local immune and inflammatory response occurs that attracts leukocytes. The antigen engulfed by the macrophages and monocytes is presented to T cells, which then becomes sensitized and activated. These cells then release cytokines and chemokines, which can cause tissue damage and may result in illnesses. Examples of illnesses resulting from type IV hypersensitivity reactions include contact dermatitis and drug hypersensitivity. Type IV reactions are further subdivided into type IVa, IVb, IVc, and IVd based on the type of T cell (CD4 T-helper type 1 and type 2 cells) involved and the cytokines/chemokines produced.[1] 

Delayed hypersensitivity plays a crucial role in our body's ability to fight various intracellular pathogens such as mycobacteria and fungi. They also play a principal role in tumor immunity and transplant rejection. Since patients with acquired immunodeficiency syndrome (AIDS) have a progressive decline in the number of CD4 cells, they also have a defective type four hypersensitivity reaction.[2][3]

Etiology

Type IV hypersensitivity reactions are, to some extent, normal physiological events that help fight infections, and dysfunction in this system can predispose to multiple opportunistic infections. Adverse events can also occur due to these reactions when an undesirable interaction between the immune system and an allergen happens. Exposure to poison ivy resulting in contact dermatitis is a classic example. Several drugs (antibiotics, anticonvulsants) can trigger type IV hypersensitivity reactions leading to drug hypersensitivity and other clinical syndromes.

Certain viral infections, when exposed to certain drugs, can trigger a reaction, such as cytomegalovirus with antibiotics, Epstein Barr virus with amoxicillin, and herpesvirus 6 with anticonvulsants. Allopurinol and, more recently, lamotrigine have been implicated in the type IV hypersensitivity reactions. Latex exposure can cause both type I and type IV hypersensitivity reactions in susceptible patients.

Epidemiology

Type four hypersensitivity is a common disorder among susceptible individuals. For example, the prevalence of contact hypersensitivity is around 1%-6% among the population. Another minor subtype of type four hypersensitivity reaction is called drug allergy. Drug allergy is sometimes considered a distinct disease and constitutes around one-seventh of the side effects of drugs. 2%-3% of hospitalized patients show allergic reactions on their skin. In Western Europe, epidemiological research shows the relationship between atopy in healthy people and purified protein derivative (PPD) response. A large cross-sectional study done in Sweden shows PPD reactions of more than 3 millimeters, which is somewhat more frequent in patients with atopy than the normal population (15.1% vs. 14.7%). However, PPD responses with a result of more than 10 millimeters were found to be 1.4% in normal children vs. 1.2% in the allergic population.[1][4][5]

Pathophysiology

The pathophysiology of type four hypersensitivity depends on the underlying cause. For instance, the granulomatous disease occurs when T cells are stimulated by antigen-presenting cells that are unable to destroy engulfed antigens. Afterward, antigen-presenting cells become giant multinucleated cells, and for this to be done, a lot of cytokines are secreted, including interleukin-2 (IL-2) and tumor necrosis factor-alpha, and beta. Another mechanism occurs in contact dermatitis when irritants or antigens are applied to the skin this will cause an inflammatory reaction mediated by over-expression of ICAM-1, VCAM-1, ELAM-1. Drug hypersensitivity occurs when various drug particles bind to a T cell receptor, even if not metabolized by antigen-presenting cells nor presented by major histocompatibility complex molecules.[6][7][8]

There are three subtypes of Type IV hypersensitivity: Contact dermatitis, tuberculin-type hypersensitivity, and granulomatous-type hypersensitivity.

Contact hypersensitivity dermatitis occurs when haptens, which are considered as exogenous antigens, penetrate the skin with proximity to epidermal and dermal cells, resulting in an inflammatory reaction. Dermal dendritic cells and Langerhans cells play an important role in antigen presentation and sensitization of these haptens to CD4 and CD8 T-cell lymphocytes. The latter secrete cytokines and other enzymes to recruit other immune cells to the site of hapten exposure. Additionally, keratinocytes help in recruiting immune cells by secreting other groups of cytokines such as IL-and IL-8. This results in inflammation of the skin with swelling, itchiness, and pain.

Tuberculin-type hypersensitivity can be seen after intradermal injection of purified protein derivative (PPD) called tuberculin (product of tuberculosis bacillus), that produces measurable local induration and swelling, typically measured in millimeters between 48 to 72 hours after the injection. This local reaction indicates the presence of type four hypersensitivity. The tuberculin test is a validated method to diagnose tuberculosis infection, even if latent.

Granulomatous-type hypersensitivity can occur in response to a variety of antigens. Macrophages that engulfed antigens are unable to destroy them and recruit several more macrophages to the site of these antigens. A collection of macrophages filled with intracellular antigens is termed granuloma. One example of granulomatous-type hypersensitivity is sarcoidosis disease, which is a systemic granulomatous disease of unknown cause, with a wide variety of clinical presentations. Sometimes, sarcoidosis is called reduced type four hypersensitivity due to the slow progression of this disease.[9][10][11][12][13][14][15]

Histopathology

After the injection of tuberculin in the skin, immune cells will infiltrate in the dermis layer of the skin, and these immune cells can be seen after staining with hematoxylin and eosin. In the granulomatous-type of hypersensitivity, a biopsy of a granuloma can show caseous necrosis surrounded by multinucleated giant cells consisting of macrophages and epithelioid cells. These granulomas are usually surrounded by lymphocytes and other immune cells. In contact hypersensitivity, mononuclear cells migrate within the epidermis and dermis. When a biopsy is taken from the skin, microvesicles can be seen between the dermis and epidermis. These microvesicles are caused as a result of edema in the skin. In irritant contact dermatitis, neutrophils are usually observed in the biopsies taken from the epidermis.[8][16]

History and Physical

The clinical features associated with type IV hypersensitivity are variable and categorized into distinct clinical conditions, each with their own unique features.

Contact dermatitis occurs after the skin is exposed to an allergen (topical medication, poison ivy) and, over a period of time, develops into a very erythematous pruritic rash, often with swelling and edema progressing to vesicles and bullae. Some of these vesicles and bullae can rupture with subsequent crust formation. When the reaction is prolonged with lichenification and scaling, the condition can be termed as subacute or chronic contact dermatitis. Some of the agents implicated in the development of contact dermatitis include gloves, clothing, acrylics, preservatives, and an array of industrial chemicals. It is, therefore, prudent to ask the patients about their occupations, hobbies, and daily activities. 

Granulomatous-type hypersensitivity can be seen in tuberculosis and sarcoidosis. Sarcoidosis is considered as a granulomatosis entity with unknown cause, with a systemic involvement of any organ in the body. It results in the formation of granulomas by the immune system in affected organs. The most frequent organs to be affected in sarcoidosis are the lymphatic system, especially in the mediastinum, lungs, eyes, and skin. In 20% to 50% of the cases, ophthalmic involvement is found. Moreover, up to 30% of patients can present with non-specific symptoms, such as weakness, weight loss, or fever. When the lungs are affected, patients usually present with shortness of breath, breathing difficulty, dry cough, and chest pain. Occasionally, these symptoms progress to pulmonary fibrosis, with a progressive decline in pulmonary functions.[16][17]

Acute generalized exanthematous pustulosis (AGEP) is a rare drug reaction presenting as generalized pustular rash manifesting within 24 hours after exposure to an offending drug.

Drug fever: Certain drugs such as trimethoprim-sulfamethoxazole or tetracyclines can cause fever as the only manifestation, and certain conditions appear to increase this susceptibility when exposed to certain drugs. Patients with acute human immunodeficiency virus (HIV) infection are more prone to get drug fever when treated with antiretroviral therapy.

Stevens-Johnson syndrome/toxic epidermal necrolysis: These are life-threatening conditions that present with severe skin and mucosal necrosis with fluid losses and can present with hypovolemic shock. There is severe blistering of the skin with pain, sloughing of the epidermis that resembles a third-degree burn. Commonly implicated agents are nonsteroidal anti-inflammatory drugs (NSAIDs), anticonvulsants, and sulfa drugs. 

Drug-induced hypersensitivity syndrome (DiHS) is another severe drug-induced type IV hypersensitivity reaction presenting with rash, fever, and multiorgan involvement, particularly the heart, lungs, liver, and kidneys.

Evaluation

The diagnosis of contact dermatitis can be made by clinical features alone, and in rare instances, a skin biopsy may be needed. To identify the offending allergen, skin patch tests may be helpful. The patients are usually tested with a wide variety of allergens and antigens to evaluate the skin response. Occasionally, immunological tests such as a complete blood count with differential, CD4, CD8, and radioallergosorbent tests may be helpful.  

If tuberculosis is suspected, tuberculin can be injected intradermally, and according to the size of the reaction that results, the patient can be diagnosed with tuberculosis. If positive, a chest Xray is warranted. In granulomatous disease such as sarcoidosis, chest Xray, lymph node biopsy, elevated serum angiotensin-converting enzyme level (not diagnostic), or testing the salivary glands or mediastinum for gallium fixation can be considered.[4][18][19][20]

Treatment / Management

Treatment of type IV hypersensitivity depends on the clinical condition that resulted from this reaction.

Contact dermatitis: Removing the offending agent is the most crucial aspect of managing this condition. The severity of the skin condition dictates the type of therapy, which would almost always include topical steroids, titrating the strength of the steroid to the severity of the dermatitis.

For Steven Johnson syndrome/toxic epidermolysis, aggressive life-saving therapy would be required, including admission to an intensive care unit, optimal fluid therapy, antibiotics if there is a secondary infection, and systemic corticosteroids.

For granulomatous conditions, therapy depends on the type of clinical condition. In both systemic and ocular sarcoidosis, steroid therapy is the standard treatment. In addition to steroids, methotrexate has shown efficacy in pulmonary sarcoidosis. In Crohn's disease, anti-tumor necrosis factor (TNF) monoclonal antibodies can be used as an effective way to manage the disease. In schistosomiasis, praziquantel can be used. Once the tuberculin test has revealed a positive result, the treatment of tuberculosis must be started, and one of the commonest regimens is to give rifampin, isoniazid, pyrazinamide, and ethambutol.[17][19][21][22][23][24]

Differential Diagnosis

Viral exanthems and certain bacterial infections can present with maculopapular rashes that can appear similar to contact dermatitis. The exposure to an allergen and the temporal relationship to the offending agent can help differentiate these conditions. 

Sarcoidosis can present with pulmonary symptoms, and a chest Xray can reveal hilar lymphadenopathy. This presentation, sometimes, can be similar to tuberculosis. However, the treatment is completely different antibiotics are prescribed in tuberculosis, while steroid therapy is needed in sarcoidosis.[25]

Prognosis

Prognosis depends on the manifested clinical condition. Most contact dermatitis resolves with generally no sequelae. Studies show that up to 40% percent of patients who avoid the allergen had not experienced any more dermatitis. 

New acute sarcoidosis is more common among Whites than Blacks, and with a higher chance of spontaneous remission within two years. This remission occurs in two-thirds of patients. However, the rest of them usually progress to chronic sarcoidosis, which usually presents with exacerbations and remissions. Mortality in sarcoidosis is usually attributed to respiratory failure and can be up to 5% of patients. In tuberculosis, the prognosis is favorable with early diagnosis and treatment with antimicrobials.[17][20]

Complications

Granulomatous diseases can affect any organ in the body, but for each type of granulomatous disease, there are common organs that are usually affected. For instance, sarcoidosis disease usually affects the lungs, eyes, and kidneys, resulting in pneumonia, lung fibrosis, pulmonary failure, cataracts, glaucoma, and kidney failure. In addition to the lungs, tuberculosis usually affects the vertebra and joints, resulting in back pain, joint stiffness, and arthritis.

In contact dermatitis, auto-eczematization can occur, which is a systemic flare of skin inflammation after the second exposure to a specific allergen. This skin inflammation disrupts the normal skin, which is the most important barrier in our body. Disruption of the skin can result in an increased risk of secondary infection of the skin by bacteria and other infectious organisms.[26] Severe Steven Johnson syndrome/toxic epidermal necrolysis can result in permanent skin scarring.

Deterrence and Patient Education

Early recognition and awareness of the occurrence of type IV hypersensitivity reactions can help patients manage their condition optimally. Patients should be educated about the common offending agents that they are susceptible to and strategies to avoid that exposure. Once the reaction occurs, prompt removal of the agent and initiation of anti-inflammatory therapy can shorten the discomfort and reduce the duration of illness.

Enhancing Healthcare Team Outcomes

Type IV hypersensitivity reaction can occur due to a wide variety of reasons affecting multiple organs, depending on individual susceptibility. Coordination of care between primary clinicians and specialists (allergists/infectious disease) can help accomplish optimal outcomes for these patients.