Molecular mechanisms of allergic disease.

Abstract: Interaction of allergen with T-cells is associated with patterns of cytokine release by immunocompetent cells characterized as T-helper Th1 or Th2 T-immune responses. The Th2 pattern of inflammation induced by this cytokine release is associated with allergic diseases. The molecular mechanisms underlying allergic inflammation are the signals for immunoglobulin (Ig) E production and the activation of mast cells and eosinophils. Data suggesting that environmental exposure may play a role in the induction of the Th2 pattern of inflammation has led to the development of the "hygiene hypothesis." Knowledge of the mechanisms of allergic inflammation has allowed the development of specific pharmacologic intervention to include 1) antibodies to IgE, 2) therapy tailored to regulate IgE production, and 3) modulation of cytokine release and function. Knowledge of the role of transcription factors in regulating gene activity as it relates to allergic inflammation is expanding and may also provide future targets for pharmacologic intervention.

Key Words: eosinophil, hygiene hypothesis, immunoglobulin E, mast cell, transcription factor

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Epidemiologic studies reflect an increasing prevalence of the allergic diseases atopic dermatitis (AD), allergic rhinitis, and asthma. A British study of school children with AD showed the prevalence of AD rose from 5.1% in 1946 to 12.2% in 1970. (1) The prevalence of rhinoconjunctivitis in 13-to 14-year-old children in 1997 varied from 1.4 to 39.7%, with the lowest prevalence in eastern Europe and south Asia and the highest in Canada, Finland, Spain, the United Kingdom, and the United States. (2) The U.S. Centers for Disease Control and Prevention reported a 73.9% increase in self-reported asthma during the period 1980 to 1996. (3) An estimated 14.6 million people in the United States reported asthma during the previous 12 months in 1996 alone. (3) Although the cause of this increase in allergic disease is unclear, the result in a given patient is a stereotyped and characteristic pattern of allergic inflammation in the airways, skin, and elsewhere.

The increasing incidence of allergic diseases has resulted in an increased burden of these disorders on the health care system. This review examines the current understanding of the molecular mechanisms of allergic inflammation and includes a discussion of the roles of T-lymphocytes, immunoglobulin (Ig) E, mast cells, and eosinophils in the pathogenesis of allergic disease. Although previous pharmacologic therapy focused on treating the symptoms of allergic disease, new therapies target the specific immunologic mechanisms of allergic inflammation. A basic understanding of the mechanisms of allergic inflammation is required to understand the rationale for these new pharmaceuticals.

T-Cell Responses in Allergic Disease

Cytokine Profile Associated with the T-helper 2 Allergic Phenotype

T-helper (Th) lymphocytes develop into either Th1 or Th2 cells and are classified on the basis of their cytokine production profile. The proteins synthesized and secreted by T-cells are referred to as cytokines. Some cytokines function as interleukins (ILs). Th1 cells produce IL-2, IL-12, tumor necrosis factor (TNF)-[beta], and interferon (IFN)-[gamma], and are involved in the elimination of intracellular pathogens. Th2 cells produce granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-4, IL-5, IL-6, and IL-13, that promote, among other things, allergic inflammation and disease mediated by IgE antibodies. Th1 and Th2 cytokine release facilitates further production of their respective cytokine repertoire, controls the development of their respective subset, and inhibits the development of the opposite subset. The expansion of Th2 cells plays a central role in the development of the allergic response, and the presence of Th2 cytokines modulates the balance of Th1/Th2 cells. The Th1-type response is promoted by exposure to inducers of IFN-[gamma], such as endotoxin. IFN-[gamma] also inhibits the Th2 response by inhibiting both IgE synthesis and the expression of the IL-4 receptor on T-cells. The potential of Th1 inducers like endotoxin to mitigate allergy is consistent with the "hygiene hypothesis."

The Hygiene Hypothesis

There is a lower prevalence of allergic disorders among children raised in rural areas versus those in urban areas in developing countries and among people who have experienced significant infections of the respiratory tract versus those who have not. (4) These findings have led to the hygiene hypothesis. The hygiene hypothesis postulates that improvements in public hygiene have lowered the prevalence of serious infections and increased the likelihood of asthma and allergy. Investigators have examined the role of environmental exposure to endotoxin, a lipopolysaccharide found on the outer membrane of Gram-negative bacteria as a modulator of the T-cell immune response. Available data suggest that a lack of endotoxin exposure is associated with a higher prevalence of atopic disease. (4)

Transcription Factors and Allergic Responses

Cytokines secreted by T-cells regulate gene activation through transcription factors. A transcription factor is a protein that directs accurate transcription of downstream genes and controls the rate of initiation of gene transcription. There are many transcription factors that play key roles in the regulation of the Th1/Th2 immune response (Table 1). IL-4, one of the most important cytokines in the allergic response, regulates gene expression through transcription factors.

IL-4, a glycoprotein produced by Th2 cells, natural killer cells, mast cells, and basophils, promotes Th2 development. IL-4 induces Th2 cell development through the transcription factor signal transducer and activator of transcription (STAT). (5) STAT6 is a member of a family of transcription factors that control genes involved in allergic responses and is expressed in the bronchial epithelium of severe asthmatics. (6) Lymphocytes from mice deficient in STAT6 fail to proliferate in response to IL-4 and demonstrate a lack of IgE class switching. (7), (8) Knowledge of transcription factors offers opportunities for new drug therapy for allergic disease.

The Role of IgE

IgE is the immunoglobulin with the lowest concentration (0-0.002 mg/ml) in normal serum, 100,000-fold less than IgG. (9) Unbound, free IgE has a half-life of 2 to 3 days, whereas IgE bound to mast cells in the respiratory tract and skin has a half-life of several weeks. IgE is composed of two heavy chains and two light chains (Fig. 1). Each heavy chain has four constant region domains (C[euro]1-C[euro]4). The constant region domain C[euro]3 is the major site of interaction with the IgE receptor, and it is this interaction that initiates the cascade of events that characterizes the allergic response. (9)

[FIGURE 1 OMITTED]

Molecular Signals for the Isotype Switch Necessary for IgE Production

The mechanism for IgE production begins when allergen is internalized by antigen-presenting cells such as macrophages, activated T-lymphocytes, and B-lymphocytes. After antigen processing within the antigen-presenting cell, peptide fragments are displayed on the cell surface of these cells in association with major histocompatibility complex class II molecules. Th2 lymphocytes recognize this complex through the T-cell receptor. This leads to the release of IL-4 and induces the expression of the CD40 ligand on the surface of the T-cell (Fig. 2).

For B-cells to switch from IgM- to IgE-bearing cells and mature into IgE-producing plasma cells (isotype switching), B-cells require two signals. The IL-4 secreted by Th2 cells provides the first signal required for B-cells to differentiate into IgE-secreting plasma cells. When it binds to the IL-4 receptor on the B-cell surface, it stimulates transcription of a gene encoding the constant region domains of the IgE heavy chain. (10) The second signal is provided by the interaction of the CD40 ligand on the surface of T-cells with the CD40 receptor on the B-cell.

Mast Cell Mediator Release and Allergic Reaction

IgE binds to the high-affinity IgE receptor (Fc[euro]RI) on mast cells, basophils, dendritic cells, eosinophils; and to low-affinity IgE receptors (CD23 or Fc[euro]RII) on monocytes, macrophages, and lymphocytes. (11) The release of mediators of immediate hypersensitivity in a sensitized patient is initiated when allergen binds to the IgE-Fc[euro]RI complex on the surfaces of mast cells (Fig. 3). This interaction results in the release of both preformed and newly synthesized inflammatory mediators.

[FIGURE 2 OMITTED]

IgE may upregulate Fc[euro]RI expression in mast cells, which permits the mast cells to be activated with lower concentrations of specific allergen. (12) After persistent allergen exposure, mast cells release larger amounts of mediators and cytokines. (13) These mast cell cytokines include IL-4, which promotes IgE production, a positive feedback. Production of vascular permeability factor (VPF)/vascular endothelial cell growth factor, important in enhancing vascular permeability, is also increased in mast cells that have undergone IgE-dependent upregulation of their surface expression of Fc[euro]RI. (13)

Mast cells develop from a population of [CD34.sup.+] hematopoietic progenitor cells and then mature in peripheral tissues. They reside in connective tissue adjacent to blood vessels and beneath epithelial surfaces. (14), (15) Increased numbers of mast cells are present in tissues affected by chronic inflammation. (14), (15)

[FIGURE 3 OMITTED]

Mast cells contain an array of preformed and stored mediators that are released on activation. Mast cells degranulate when IgE antibodies bound to mast cells are cross-linked by allergen. At the site of allergen exposure, these proinflammatory mediators cause mucus secretion, airway smooth muscle contraction, and mucosal edema. Preformed mediators stored in cytoplasmic granules include histamine, tryptase, proteoglycans, chymase, carboxypeptidase A, and heparin. These mediators are involved in the immediate clinical responses of vasodilation, edema, bronchoconstriction, and itching. Mast cells contain preformed stores of the cytokines such as TNF-[alpha] and VPF, as well as many other cytokines including IL-2, IL-3, IL-4, IL-13, GM-CSF, and chemokines. (15) Chemokines are a family of low-molecular-weight chemotactic cytokines. Mast cells also release newly synthesized lipid mediators prostaglandin [D.sub.2] and leukotriene C4, as well as cytokines.

Acute allergic reactions develop within minutes of allergen exposure; late-phase inflammatory reactions also occur in the skin and airway. There is evidence that a mast cell-leukocyte cytokine cascade initiates the late-phase component of the allergic response and contributes to chronic allergic inflammation through effects of fibroblasts, vascular endothelial cells, and therefore tissue remodeling. (15) Cytokines such as TNF-[alpha] and VPF/vascular endothelial cell growth factor may contribute to a process of tissue remodeling in the chronic allergic response. (15)

Eosinophils

Eosinophil Development

Eosinophils develop in the bone marrow from [CD34.sup.+] progenitor cells under stimulation of IL-3, IL-5, and GM-CSF. (16) The genes encoding these cytokines are closely linked and are found on chromosome 5. Although all three cytokines promote eosinophilopoiesis, IL-5 uniquely promotes the development and differentiation of eosinophils. IL-5, a product of Th2 cells, also plays an important role in the mobilization of eosinophils from the bone marrow and into the bloodstream. Mature eosinophils first detach from the bone marrow extracellular matrix and then migrate across the bone marrow sinus endothelium. The eosinophils are then released from the endothelium and into the bloodstream or are retained in the medullary cavity of the marrow. IL-5 can also induce the rapid release of pooled eosinophils from the bone marrow. Eosinophils circulate in the peripheral blood with a normal half-life of 8 to 18 hours. Once they have migrated into peripheral tissues, they persist for several days. (14)

Eosinophil Migration

The migration of eosinophils from the peripheral blood into tissues is a multistep process mediated by cytokines, chemoattractants, selectins, and integrins (Fig. 4). Integrins are a family of cell surface proteins that mediate cell-to-cell and cell-to-extracellular matrix interactions. Both neutrophils and eosinophils migrate into peripheral tissues by rolling along, adhering to, and then passing between endothelial cells. The rolling of eosinophils is primarily mediated by E-selectin. Eosinophils undergo cellular activation after being exposed to chemoattractants such as the chemokines, platelet-activating factor (PAF), and eotaxin, as well as IL-5. (17) Once activated, eosinophils are able to firmly adhere to the endothelium through the interaction of integrins with their corresponding receptors on the endothelial surface. The integrin family includes the CD18 family and the very late antigen (VLA)-4 molecules. Integrins are normally expressed on eosinophil cell surfaces but are stimulated to an increased level of surface expression when the cell is activated by chemoattractants like PAF and chemokines. (16)

[FIGURE 4 OMITTED]

When compared with neutrophils, eosinophils have both shared and distinct adhesion pathways. CD18 interaction with intercellular adhesion molecule (ICAM)-1 on endothelial cells is a shared pathway between neutrophil and eosinophil adhesion. ICAM-1 expression on endothelial cell surfaces is induced by the proinflammatory cytokines IL-1 and TNF-[alpha]. Unique to eosinophils is the VLA-4 interaction with vascular cell adhesion molecule (VCAM)-1. VCAM-1 is primarily induced by IL-4, and increased levels of both ICAM-1 and VCAM-1 have been found in tissue biopsy specimens of patients with allergic disorders. (17)

Chemoattractant molecules locally mediate the migration of eosinophils into tissues. These molecules include lipid mediators such as PAF, as well as various chemokines. They include RANTES (regulated on activation, normal T-cell expressed and presumably secreted), macrophage chemotactic protein (MCP)-2, MCP-3, MCP-4, eotaxin-1, and eotaxin-2. Both eotaxin-1 and eotaxin-2 are chemokines specific for eosinophils. Most chemokines interact with eosinophils through the chemokines receptor (CCR)-3, and eosinophil response to chemokines can be blocked by antibodies to CCR-3. (18)

Eosinophil Products and Their Roles in Allergic Inflammation

Once they are in local tissues, eosinophils release toxic inflammatory mediators. Mediators stored in granules include major basic protein (MBP), eosinophil peroxidase, and eosinophil cationic protein. MBP increases smooth muscle reactivity, and MBP, eosinophil peroxidase, and eosinophil cationic protein are known to have cytotoxic effects on respiratory epithelium. Eosinophils also synthesize and release lipid mediators such as leukotriene C4 that cause increased vascular permeability and stimulate smooth muscle contraction. Other eosinophil products that contribute to the allergic inflammation are PAF, GM-CSF, IL-5, RANTES, and eotaxin. Eosinophils and basophils have receptors for IL-3, IL-5, and GM-CSF both on their bone marrow precursors and on circulating cells. In the absence of these cytokines, survival is limited to less than 48 hours. In their presence, they may persist for weeks. (17)

Early and Late-phase Allergic Inflammation

The collaboration of mast cells and eosinophils in IgE-mediated inflammation clinically results in early and late-phase allergic responses. Individuals genetically predisposed to produce IgE to allergen do so after initial exposure. Then, IgE fixes to their mast cells and they are thus "sensitized." In some of these patients, reexposure leads to release and an allergic reaction. Immediate allergen reactions begin when allergen comes into contact with allergen-specific IgE on the mast cell surface. These activated mast cells then degranulate and release various proinflammatory mediators. Histologic sections of bronchial biopsy specimens from patients with asthma who died as a result of an acute asthma exacerbation reveal sloughing of the surface epithelium, a thickened basement membrane, an enlargement of bronchial smooth muscle, mucous plugging, and an infiltration of the mucosa by inflammatory cells. (19)

The late-phase response occurs from 6 to 9 hours later and involves the recruitment and activation of eosinophils, neutrophils, T-cells, and macrophages to the site of allergen challenge. The release of cytokines by mast cells during the early response is likely one of the initial triggers for recruitment of eosinophils and T-cells. The subsequent release of Th2 cytokines by recruited T-cells serves to augment inflammatory cell recruitment. Eosinophils transmigrate from the peripheral circulation into tissues through specific homing adhesion molecules and migrate to the site of inflammation using chemokine gradients. When they arrive, they undergo degranulation and release mediators of inflammation. Histopathologic changes observed in the airways of asthmatics 24 hours after allergen challenge reveal denuded epithelial surfaces, thickened basement membranes, and mucosa containing degranulated eosinophils. (19)

Potential Therapeutic Targets

Anti-IgE

Current pharmacologic treatment of allergic disorders is largely nonspecific and intervenes at the later stages of the allergic cascade. Advances in the pathophysiologic mechanisms underlying the allergic disorders have allowed for the development of specific pharmacologic therapies. One such newly developed drug is omalizumab (anti-IgE), a recombinant humanized antibody that binds IgE. Omalizumab binds only to the C[euro]3 region of circulating IgE and therefore does not cause degranulation of mast cells and basophils with IgE already bound to the cell surface. Omalizumab is specific for IgE and does not bind to circulating IgG or IgA.

Preclinical trials revealed anti-IgE therapy resulted in decreased serum IgE levels in a dose-dependent manner as soon as 5 minutes after IV injection and within 24 hours of SC injection. (20) Over time, IgE levels return to pretreatment levels as IgE and anti-IgE complexes circulate. Clinical trials have demonstrated that omalizumab has been shown to reduce the number of asthma exacerbations in patients with moderate to severe asthma who are taking corticosteroids. (21) Therapy also resulted in improvement in symptom scores and a reduction in the requirement for steroid medications. Early clinical studies in patients with seasonal allergic rhinitis have also shown effectiveness in reducing symptom scores and the need for antihistamine therapy. (22) In a study of patients with peanut allergy, anti-IgE administered SC significantly increased the threshold of sensitivity to peanut antigen. (17) Although omalizumab is now available for the treatment of asthma, there are no practice guidelines incorporating this novel agent into the management of allergic disorders, and studies are needed to further delineate the role of anti-IgE in the treatment of asthma, seasonal allergic rhinitis, and food allergy.

Regulation of IgE Production

Another potential therapeutic target is the upstream regulation of IgE production. This approach would inhibit IgE class switching by interfering with the interaction between IL-4 and the IL-4 receptor. This interaction provides the first of two signals needed for initiation of allergic inflammation. Without this signal, B-cells would not be stimulated to differentiate into IgE-secreting plasma cells. Eosinophil adhesion would also be adversely affected, and IL-4 and IL-5 production would be downregulated. Therefore, blockade of the effects of IL-4 would not only interfere with IgE class switching but would also inhibit the Th2 cell and its function in the allergic response.

Soluble recombinant IL-4 receptor (altrakincept) has been produced as a therapeutic treatment. It inactivates naturally occurring IL-4 and has been found to have some steroid-sparing effects in asthma. (22) Antibodies have also been developed that target eosinophil recruitment and activation, such as antibodies against IL-5 and CCR-3. (18) Yet another target is eosinophil adhesion to endothelial surfaces through the interaction on VLA-4 with VCAM-1. (16)

Cytokine Modulation

Another approach attempts to regulate the synthesis of IgE by modulating the cytokines involved in the production of IgE. IgE synthesis can be suppressed by the cytokines IFN-[gamma] and IL-12. IL-12 and IFN-[gamma] both inhibit Th2 cytokine production. IL-12 has been shown in experimental models to inhibit the production of IL-4 and IL-5 and to reduce pulmonary eosinophilia after exposure to allergen. (16) IFN-[gamma] has minimal effects on IgE levels, and this may be possibly explained by the fact that B-cells that have already switched to IgE synthesis are no longer responsive to inhibition by IFN-[gamma]. (23) Yet another approach would be regulation of cytokine production by modulation of transcription factors. GATA-3 is a transcription factor that controls expression of IL-4, IL-5, and IL-13. GATA-deficient mice have decreased levels of IgE and eosinophilia after allergen challenge. (24)

Key Points

* The production of interleukin (IL)-4 and IL-5 by helper-inducer lymphocytes characterizes allergic inflammation.

* For B-cells to undergo isotype switching necessary to produce immunoglobulin E, they must receive two signals: 1) stimulation of the IL-4 receptor by IL-4 and 2) the interaction of the CD40 receptor with its corresponding CD40 ligand on the surface of T-cells.

* The mediators of immediate hypersensitivity are released in the sensitized patient when allergen binds to the immunoglobulin E-Fc[euro] receptor I complex on the surface of mast cells.

* At the site of allergen exposure, inflammatory mediators of the early and late-phase allergic responses cause mucus secretion, airway smooth muscle contraction, and mucosal edema.

* Knowledge of the mechanisms of allergic inflammation allows for the development of targeted pharmacologic therapy.

Table 1. Transcription factors involved in the

regulation of Th1/Th2 responses (a)

                            Effects on

                          Th2        Th1
Transcription factors  phenotype  phenotype

STAT4                                 +

STAT6                      +

BCL-6                      -

T-bet                      -          +

GATA-3                     +          -

C-maf                      +

NF-Atc                     +

NF-ATp/NF-AT4              -

(a) Th, T-helper; STAT, signal transducer and activator of
transcription. From, Escoubet-Lozach L, Glass CK. Wasserman SI. The role
of transcription factors in allergic inflammation. J Allergy Clin
Immunol 2002;110:553-564. (5)

From the Departments of Medicine and Pediatrics, University of Mississippi Medical Center, Jackson, MS.

Reprint requests to Richard D. deShazo, MD, Department of Pediatrics, University of Mississippi Medical Center, 2500 N. State Street, Jackson, MS 39216. Email: [e-mail]

Accepted August 19, 2003.

Copyright [c] 2003 by The Southern Medical Association

0038-4348/03/9611-1049

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Daniel Venarske, MD, and Richard D. deShazo, MD