IMMUNOLOGY - Roitt I. - Mir 2000

Chapter 26. Hypersensitivity - Type IV

■ Three types of type IV hypersensitivity reactions are known: contact, tuberculin, and granulomatous.

■ A hapten applied to the Skin is taken up and processed by Langerhans Cells; these same cells present the hapten to antigen-specific T cells.

■ Cytokines produced by immunocompetent skin cells (e.g., keratinocytes, Langerhans cells, or T cells) drive the recruitment of antigen-nonspecific T cells and macrophages in the reaction.

■ The tuberculin-type hypersensitivity reaction can be used as a diagnostic test for the presence of many infectious agents.

■ Granulomatous reactions are characterized by a specific balance between protective Immunity against an insoluble antigen and T Cell-mediated tissue damage. A classic example of this balance is the tuberculoid form of leprosy.

■ Antigen persistence leads to the differentiation of macrophages into epithelioid cells and their fusion to form giant cells. This pathological process, termed a granulomatous reaction, results in tissue damage.

■ Granuloma formation is associated with T cell-mediated macrophage activation and is dependent on TNF.

According to the Gell and Coombs Classification, type IV (delayed-type) hypersensitivity comprises reactions that manifest no earlier than 12 hours and are mediated by cellular rather than humoral immune mechanisms. However, this classification does not fit certain hypersensitivity reactions. For example, late-phase reactions, which peak 12–24 hours after allergen contact, are mediated predominantly by IgE, although T helper cells also participate, meaning their mechanism is complex. Other reactions (such as Jones-Mote hypersensitivity, which resembles cutaneous basophil hypersensitivity in guinea pigs) were also previously classified as type IV, although their mechanisms and clinical significance remain unclear; they are not discussed in this chapter.

Unlike Other forms of hypersensitivity, type IV reactions can be transferred from a sensitized animal to a non-sensitized one not by serum, but by T cells (Th1 cells in the mouse). These reactions are undoubtedly underpinned by the mechanisms of protective cellular immunity, yet a full correlation between protective immunity and type IV hypersensitivity is not always observed. The T cells responsible for the delayed reaction are specifically sensitized by the antigen during prior contact and act by recruiting other cell types to the reaction site.

Three variants of type IV hypersensitivity reactions are recognized (Fig. 26.1). Contact and tuberculin reactions develop within 72 hours of antigen exposure, whereas the granulomatous reaction takes 21–28 days; granulomas form As a result of macrophage accumulation and proliferation and can persist for weeks. This form of type IV hypersensitivity causes the most severe clinical consequences. It should be noted that the same antigen can trigger Different types of reactions that may overlap.

These three types of delayed-type reactions were originally distinguished by The Nature of the manifestations that occur upon topical application or intradermal injection of the antigen. The severity of the reaction in animals is usually assessed by measuring the thickness of the affected skin area. Various systemic immune responses may also develop following the local reaction.

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Fig. 26.1. Contact and tuberculin forms of hypersensitivity share a similar temporal dynamic, peaking at 48–72 hours. In some cases (e.g., exposure to an insoluble antigen), a granulomatous reaction also develops after 21–28 days (e.g., in the lepromin skin test).

CONTACT HYPERSENSITIVITY

Contact hypersensitivity is characterized by an eczematous reaction at the site of antigen exposure (Fig. 26.2). It frequently results from contact with substances such as nickel, chromates, rubber industry catalysts, and pentadecylcatechol (found in poison ivy). Eczema can also occur upon contact with toxic irritants without the involvement of hypersensitivity mechanisms. Although the initial reactions in these two scenarios differ, the immunological events that develop following exposure to irritants and allergens are similar.

Fig. 26.2. Clinical manifestations and skin test results in contact hypersensitivity. 1. Eczematous lesions on the wrist caused by a nickel watch buckle. 2. Suspected allergy can be confirmed by applying potential allergens (at an adequate concentration and in a suitable vehicle) to the skin of the inner forearm (patch test). A positive reaction is indicated by the appearance of a localized patch of eczema at the site of allergen application within 2–4 days.

The immunologically active Components of the agents listed above are termed haptens. Haptens are not antigenic on their own (their molecules are too small for this—molecular mass often does not even reach 1 kDa), but upon penetrating the epidermis and binding (most commonly covalently) to the body's own Proteins, they acquire antigenic activity. The sensitizing potential of haptens is difficult to predict from their Structure, although there is some correlation between The amount of carrier-bound hapten and its ability to penetrate the skin. Certain contact allergens contain unsaturated carbon bonds and are readily oxidized. Some haptens, such as 1-chloro-2,4-dinitrobenzene (DNFB), sensitize almost every individual and can be used to assess cell-mediated immunity. Applied to the skin, DNFB binds to epidermal proteins via the NH₂ groups of Lysine residues.

Langerhans cells and keratinocytes play a key role in contact hypersensitivity

Langerhans cells serve as the primary antigen-presenting cells. Contact hypersensitivity is predominantly an epidermal reaction, and the main antigen-presenting cells (APCs) in this case are Langerhans dendritic cells located in the suprabasal layer of the epidermis (Fig. 26.3). These cells are of Bone Marrow origin and express the CD1 surface marker, MHC class II Antigens, and Fc and Complement receptors (see Chapter 2). Electron Microscopy reveals that Langerhans cells contain Birbeck granules (cell-specific Organelles formed from The Cell membrane). Ultraviolet B radiation inactivates Langerhans cells, which prevents or mitigates the manifestations of contact hypersensitivity.

Fig. 26.3. A Langerhans cell.

1. These dendritic cells account for 3% of all epidermal cells. They express a range of surface markers that allow them to be visualized. In this instance, a section of normal skin is stained with Monoclonal Antibodies against CD1 (counterstained with Mayer's hemalum).

(L - Langerhans cell; K - keratinocyte.) x 312.

2. An electron micrograph of a Langerhans cell showing a characteristic Birbeck granule. This lamellar organelle features distinct central striations and is often dilated at one end. x 132,000.

In vitro, Langerhans cells act as APCs, exhibiting higher activity in this regard than monocytes. However, the exact mechanism of antigen Processing in Langerhans cells remains unclear. Antigens associated with MHC class II molecules may be internalized by these cells via receptor-mediated endocytosis involving Birbeck granules.

Keratinocytes produce a range of cytokines critical for contact hypersensitivity reactions

Keratinocytes maintain the structural integrity of the epidermis and play a central role in the immune responses of this skin layer. They can express MHC class II and ICAM-1 molecules on their membrane, as well as secrete cytokines, including IL-1, IL-3, IL-6, IL-8, GM-CSF, TNFα, TGFα, and TGFβ. Interleukin-3 is capable of activating Langerhans cells, stimulating proliferative responses, recruiting mast cells, and inducing the secretion of immunosuppressive cytokines (such as IL-10 and TGFβ) that suppress the Immune Response and induce clonal anergy (immunological unresponsiveness) in Th1 cells.

Keratinocytes are activated by numerous stimuli, including allergens and irritants. Upon activation, they produce immunoregulatory cytokines such as TNFα and GM-CSF, which in turn activate Langerhans cells. Certain antigens, including urushiol from poison ivy, directly induce The production of TNFα and IL-8.

The contact hypersensitivity reaction develops in two stages: sensitization and elicitation

Immunological memory T cells are formed during the sensitization phase. In humans, the sensitization period lasts 10–14 days. Upon penetrating the skin, a hapten binds to a protein and is subsequently taken up (internalized) by epidermal Langerhans cells. These cells then leave the epidermis and migrate via afferent Lymphatic vessels to the paracortical zone of regional Lymph Nodes. Here, they present processed hapten-protein conjugates (associated with MHC class II molecules) to CD4⁺ lymphocytes, which give rise to the pool of immunological memory cells (Fig. 26.4). Studies in humans using DNFB demonstrate that sensitization depends primarily on the hapten dose per unit of skin area, rather than the total dose or the total surface area exposed to the hapten.

Fig. 26.4. A hapten-carrier protein complex is formed in the epidermis. Langerhans cells engulf (internalize) the antigen and migrate via afferent lymphatic vessels to the paracortical area of the regional lymph node, where, acting as interdigitating cells, they present the antigen to CD4⁺ T cells.

The elicitation phase of the reaction proceeds with the participation of CD4⁺ lymphocytes and monocytes

Within a few hours following topical allergen application, a transient decrease in the number of epidermal Langerhans cells is typically observed. Subsequently, these cells present the antigen in the skin and lymph nodes. Shortly thereafter, mast cell degranulation occurs, accompanied by cytokine release. Cytokines produced by various cell types, notably macrophages—such as TNFα and IL-1—serve as potent Inducers of adhesion molecule expression by endothelial cells. The local release of these cytokines establishes a concentration gradient, signaling mononuclear cells to migrate toward the dermo-epidermal junction and the epidermis. The elicitation phase of contact hypersensitivity is illustrated in Fig. 26.5.

Fig. 26.5. Langerhans cells bearing the hapten-carrier complex (1) migrate from the epidermis into the dermis, where they present this complex to immunological memory CD4⁺ T cells (2). Activated CD4⁺ T cells release IFNγ, which induces the expression of ICAM-1 (3) and, subsequently, MHC class II molecules (4) On the surface of keratinocytes and cutaneous capillary endothelial cells, while also stimulating keratinocytes to produce proinflammatory cytokines such as IL-1, IL-6, and GM-CSF (5). These cytokines recruit antigen-nonspecific CD4⁺ T cells to the reaction site (6), which can bind to keratinocytes via ICAM-1 and MHC class II molecules. Activated macrophages are also recruited to the lesion, albeit at a later stage. From this point onward, the reaction begins to subside. Eicosanoids, such as PGE secreted by activated keratinocytes and macrophages, may participate in suppressing the reaction (7).

The earliest histological change (appearing at 4–8 h) consists of the accumulation of mononuclear cells around Skin Appendages (Nails, Hair follicles) and Blood Vessels, which subsequently infiltrate the epidermis. Macrophages invade the dermis and epidermis by 48 h. The number of cells infiltrating these skin layers peaks at 48–72 h (Fig. 26.6), with the majority of lymphocytes belonging to the CD4⁺ subset and only a few to the CD8⁺ subset. Less than 1% of the skin-infiltrating cells belong to a single clone (specifically, the clone of antigen-specific immunological memory CD4⁺ Th1 cells). T-cell recruitment occurs primarily via an antigen-independent mechanism.

Fig. 26.6. Histological appearance of tissue injury in a contact hypersensitivity reaction. Mononuclear cells (M) infiltrate the dermis and epidermis. The epidermis swells, and microvesicles (V) form as a result of edema (E). Hematoxylin and eosin stain, x 130.

The mechanisms of reactions to allergens and irritants share partial similarities

Both allergens and irritants can damage Langerhans cells and induce the release of cytokines that promote the maturation of these cells into active APCs, which then migrate to LYMPH NODES AND initiate an immune response. mRNA synthesis for TNFα, IFNγ, and GM-CSF begins as early as 30 min after topical application of an allergen or irritant, and increases tenfold within 2–4 h. Certain transcriptional changes in mRNA are triggered exclusively by hapten exposure. These include an increased level of IL-1β mRNA in Langerhans cells (within 15 min), as well as enhanced METABOLISM/31.html">Transcription of IL-1α mRNA in keratinocytes, macrophage inflammatory protein-2 (MIP-2), and interferon-inducible protein-10 (IP-10) (Fig. 26.7).

Fig. 26.7. Cytokines and Prostaglandins play a central role in the complex interactions among Langerhans cells, CD4⁺ T cells, keratinocytes, macrophages, and endothelial cells during a contact hypersensitivity reaction. Antigen presentation (1) triggers a cascade of cytokine release (2). This initially leads to the activation and proliferation of CD4⁺ T cells (3), upregulation of ICAM-1 and MHC class II molecule expression on The surface of keratinocytes and endothelial cells (4), and the recruitment of T cells and macrophages into the skin (3, 5). Subsequent production of PGE by keratinocytes and macrophages can inhibit the synthesis of IL-1 and IL-2. Resolution of the reaction is facilitated not only by PGE production, but also by the binding of activated T cells to keratinocytes, alongside enzymatic and cellular degradation of the hapten-carrier complex.

Chemical exposure of the skin can lead to the upregulation of ELAM-1 and VCAM-1 expression within 2 h, and ICAM-1 within 8 h, regardless of prior sensitization. ICAM-1 is of greater importance than ELAM-1 and VCAM-1, as this molecule serves as a Ligand for LFA-1 present on the surface of lymphoid and myeloid cells, playing a crucial role in retaining these cells within the skin. Chemotactic cytokines and the "signaling effect" of migrating Langerhans cells attract Th1 cells. Immunological memory T cells remain within cutaneous capillaries, where they can initiate reactions and accumulate independently of the presence of the specific antigen.

A number of cytokines suppress the inflammatory response

By 48–72 h, the reaction subsides; macrophages and keratinocytes secrete PGE, which inhibits the production of IL-1 and IL-2; T cells bind to activated keratinocytes, and the hapten conjugate undergoes enzymatic and cellular degradation. The resolution of the reaction is facilitated by the following mechanisms.

✵ Migration inhibitory lymphokines prevent the spread of the inflammatory response.

✵ TGF-$eta$, secreted by dermal mast cells activated by keratinocytes and lymphocytes, inhibits inflammation and blocks the proliferative effects of IL-1 and IL-2.

✵ IL-1, synthesized by keratinocytes upon contact with allergens, inhibits oxidative metabolism in macrophages and suppresses their production of proinflammatory mediators.

✵ IL-10 attenuates the expression of class II molecules and suppresses cytokine production as well as antigen-specific proliferation of Th1 cells.

✵ The involvement of external factors is also possible; for instance, UV irradiation in mice induces the synthesis of a specific inhibitor of IL-1 activity.

✵ Keratinocytes expressing class II molecules in the absence of costimulatory molecules cannot act as APCs, but, upon expressing a hapten, are capable of inducing clonal anergy in Th1 cells.