Assessing basophil functional measures during monoclonal anti-IgE therapy

Assessing the impact of therapeutic interventions on the clinical and immunologic responses of allergic subjects is a topic of extensive investigation. Available approaches include the measurement of in vivo allergen challenge responses, serologic measures, or in vitro studies of cells that participate in the allergic reaction. Several decades of work support that measures of allergen responses of IgE-bearing peripheral blood basophils can reflect clinical expression of allergic disease. In the last decade, an immune-based therapy targeting IgE, omalizumab, has emerged as an adjunct treatment for a variety of allergic diseases. This monoclonal humanized IgG antibody specifically binds circulating IgE at a region in the Fc tail that prevents IgE attachment to high affinity IgE receptor (FcεRI) bearing cell types such as tissue mast cells and blood basophils. This review focuses on methods to monitor changes of basophil allergen reactivity with a focus on omalizumab therapy and the implications for clinical disease management.     

The role of allergen-specific IgE,IgG and IgA in allergic disease

Immunoglobulin E (IgE)-mediated allergy is the most common hypersensitivity disease affecting more than 30% of the population. Exposure to even minute quantities of allergens can lead to the production of IgE antibodies in atopic individuals. This is termed allergic sensitization, which occurs mainly in early childhood. Allergen-specific IgE then binds to the high (FcεRI) and low-affinity receptors (FcεRII, also called CD23) for IgE on effector cells and antigen-presenting cells. Subsequent and repeated allergen exposure increases allergen-specific IgE levels and, by receptor cross-linking, triggers immediate release of inflammatory mediators from mast cells and basophils whereas IgE-facilitated allergen presentation perpetuates T cell–mediated allergic inflammation. Due to engagement of receptors which are highly selective for IgE, even tiny amounts of allergens can induce massive inflammation. Naturally occurring allergen-specific IgG and IgA antibodies usually recognize different epitopes on allergens compared with IgE and do not efficiently interfere with allergen-induced inflammation. However, IgG and IgA antibodies to these important IgE epitopes can be induced by allergen-specific immunotherapy or by passive immunization. These will lead to competition with IgE for binding with the allergen and prevent allergic responses. Similarly, anti-IgE treatment does the same by preventing IgE from binding to its receptor on mast cells and basophils. Here, we review the complex interplay of allergen-specific IgE, IgG and IgA and the corresponding cell receptors in allergic diseases and its relevance for diagnosis, treatment and prevention of allergy.     

Induction of basophil desensitization in physiological medium: enhancement after IgE-dependent upregulation of surface IgE binding on basophils

BACKGROUND: Although the ability of basophils to release mediators, called releasability, may be an important aspect which influences the proinflammatory role of these cells, clinical approaches aiming at the depletion of the releasability have not been established. We examined whether the desensitization procedure in Ca(2+)-containing physiological conditions can make basophils completely unresponsive to IgE-mediated stimulation, and whether basophil desensitization is affected by the surface IgE levels. METHODS: Human peripheral blood basophils were cultured with low concentrations of anti-IgE antibody or recombinant mite allergen. Following culture, cells were stimulated and their histamine release was measured. RESULTS: Culturing with mite allergen or anti-IgE antibody below threshold concentrations induced potent desensitization in basophils. The desensitizing effect of anti-IgE was dose- and time-dependent; IgE-dependent releasability was completely suppressed when basophils were incubated with a near-threshold concentration of anti-IgE for > or= 4 h. In the continuous presence of subthreshold doses of anti-IgE, basophils remained desensitized even after 3 days. Basophils which had undergone an increase in surface IgE levels after 24-hour culture with IgE demonstrated enhanced desensitization. CONCLUSIONS: Near-threshold stimulation in physiological medium can affect basophils, thereby inducing complete and sustained deprivation of releasability without triggering degranulation. Basophil desensitization is regulated by their surface IgE levels. Induction of full desensitization may represent a potentially important therapeutic strategy for IgE-mediated allergic diseases in which basophils play pathogenic roles.     

Effects of omalizumab on markers of inflammation in patients with allergic asthma

Asthma is a chronic inflammatory disease of the airways in which immunoglobulin E (IgE) plays a key role by activating a variety of inflammatory cells through interactions with FcɛRI and FcɛRII receptors. The role of IgE in allergic inflammation provided the rationale for developing omalizumab, a humanized monoclonal anti-IgE antibody, for patients with moderate-to-severe or severe allergic asthma. The reductions in circulating levels of IgE resulting from omalizumab treatment leads to reductions in FcɛRI expression on mast cells, basophils and dendritic cells. This combined effect results in attenuation of several markers of inflammation, including peripheral and bronchial tissue eosinophilia and levels of granulocyte macrophage colony stimulating factor, interleukin (IL)-2, IL-4, IL-5 and IL-13. By blocking IgE binding to its receptors and diminishing dendritic cell FcɛRI receptor expression, omalizumab may also reduce allergen presentation to T cells and the production of Th2 cytokines. The anti-inflammatory effects of omalizumab may, therefore, explain the reductions in asthma exacerbations and symptoms seen in clinical trials in patients with moderate-to-severe or severe, persistent, inadequately controlled allergic asthma.     

Role of IgG4 in histamine release from human basophil leucocytes. I. Sensitization of cells from normal donors

Several conflicting reports on the ability of IgG4 to mediate type I allergic reactions have appeared lately. We have developed a model system for testing this possibility, using passive sensitization of basophil leucocytes from normal individuals. At first, the system was optimized with regard to donor cells and anti-IgG4 antibody: among 3 normal individuals with cells showing high, medium and low responses to anti-IgE, only the high responder showed a reproducible, but low response to anti-IgG4. This response was consistent when testing anti-IgG4 from different sources, and could be potentiated by a phorbol ester TPA, although not up to the level of anti-IgE. The cells from the high responding donor and a monoclonal anti-IgG4 were selected for further studies. Serum pools from patients allergic to house dust mite (Dermatophagoides pteronyssinus) were used for passive sensitization. The pools contained allergen-specific IgE with or without concomitant IgG4 of the same specificity. The sensitized basophils reacted to anti-IgE and allergen, but to anti-IgG4 only to a small extent. However, when these serum pools were fractionated by affinity chromatography, only the IgE fractions and not the IgG4 fractions made the cells reactive towards specific allergen. It was still possible to elicit a reaction by triggering IgE with anti-IgE, whereas only a small reaction was seen, when IgG4-sensitized basophils were provoked with anti-IgG4. We conclude that IgG4 does not sensitize normal basophils.     

An immunoglobulin E-reactive chimeric human immunoglobulin G1 anti-idiotype inhibits basophil degranulation through cross-linking of FcɛRI with FcγRIIb

Background IgE binds to mast cells and basophils via its high‐affinity receptor, Fc?RI, and cross‐linking of Fc?RI‐bound IgE molecules by allergen leads to the release of allergic mediators characteristic of type I hypersensitivity reactions. Previous work has shown that cross‐linking of Fc?RI with FcγRIIb, an ITIM‐containing IgG receptor, leads to inhibition of basophil triggering. 2G10, a chimeric human IgG1 anti‐idiotype, has broad reactivity with human IgE and as such has the potential to bind simultaneously to Fc?RI‐bound IgE, via its Fab regions, and the negative regulatory receptor, FcγRIIb, via its Fc region. Objective To assess the ability of human 2G10 to inhibit anti‐IgE and allergen‐driven basophil degranulation through cross‐linking of Fc?RI‐bound IgE with FcγRIIb. Methods 2G10 was assessed for its ability to bind to FcγRIIb on transfected cells and on purified basophils. In the basophil degranulation assay, basophils were purified from peripheral blood of atopic individuals and activated with either anti‐IgE or the house dust mite allergen Der p 1, in the presence or absence of human 2G10. Basophil activation was quantified by analysis of CD63 and CD203c expression on the cell surface, and IL‐4 expression intracellularly, using flow cytometery. Results Human 2G10 was able to bind to FcγRIIb on transfected cells and on purified basophils, and induce a dose‐dependent inhibition of both anti‐IgE and Der p 1‐driven degranulation of basophils. Conclusion The inhibition of basophil degranulation by the human IgG1 anti‐idiotype 2G10 highlights the therapeutic potential of IgE‐reactive IgG antibodies in restoring basophil integrity through recruitment of the inhibitory receptor FcγRIIb.     

Omalizumab-induced reductions in mast cell Fce psilon RI expression and function

BACKGROUND: By design, omalizumab binds free IgE in the circulation and prevents its attachment to the surface of mast cells and basophils, thereby preventing them from responding to allergens. Previously, omalizumab rapidly reduced free IgE levels, as well as basophil high-affinity IgE receptors, leading to significant reductions in basophil mediator response to allergen. It is assumed that tissue mast cells are similarly altered in their Fc epsilon RI density and function. OBJECTIVE: We examined the phenotypic shift of skin mast cells in parallel to that of blood basophils in 3 subjects infused with omalizumab. METHODS: Three subjects with allergic rhinitis underwent intradermal skin test titration with house dust mite antigen at days 0, 7, 70, and 196 of omalizumab treatment. As control subjects, 5 untreated subjects with allergic rhinitis were evaluated at similar time points. All subjects underwent skin biopsy 18 to 24 hours later at the site of allergen injection. Biopsy specimens were characterized by means of immunohistochemisty for tryptase and Fc epsilon RI alpha immunoreactivity, as well other markers (CD3, CD45RO, CD68, cutaneous lymphocyte antigen, and major basic protein). RESULTS: Omalizumab recipients, but not control subjects, demonstrated reductions in Fc epsilon RI alpha immunoreactivity at days 70 and 196 in parallel with reductions in the acute wheal response to allergen. However, no reductions in tryptase-positive cells were noted at these time points. CONCLUSION: Reductions in free IgE levels by omalizumab leads to a rapid reduction in basophil Fc epsilon RI receptor expression. In contrast, the time course for the decrease of Fc epsilon RI expression in skin mast cells is slower and associated with decreased acute allergen wheal size.     

In vitro basophil histamine-releasing activity of circulating IgG1 and IgG4 autoanti-IgE antibodies from asthma patients and the demonstration that anti-IgE modulates allergen-induced basophil activation

In this study we have examined the relationship between the in vitro basophil histamine-releasing activity of human IgG anti-IgE, isolated as euglobulin fractions from sera of asthmatic patients, and its IgG1/IgG4 subclass distribution. In particular, we have investigated whether IgG anti-IgE modulates allergen-induced basophil activation. The study has revealed that only a small proportion of IgG anti-IgE samples triggered histamine release from basophils of an asthmatic individual (4/21; 19%), a hay fever sufferer (4/10; 40%) and a healthy person (7/21; 33%). The basophil histamine-releasing activity of IgG anti-IgE did not seem to be determined by the IgG1/IgG4 subclass composition of the IgG anti-IgE preparation used. Furthermore, we have demonstrated that autoanti-IgE antibodies modulate allergen-induced basophil histamine release. The three modulatory effects exerted by IgG anti-IgE antibodies on allergen-triggered basophil activation (i.e. additive, synergistic and blocking) were not dependent on the subclass nature of IgG anti-IgE or the use of histamine-releasing anti-IgE preparations. Our data suggest that IgG anti-IgE antibodies in asthma patients may consist of two functionally distinct subpopulations: those which up-regulate (pro-allergic) and those which down-regulate (anti-allergic) the allergic release of mediators from mast cells and basophils.     

Anti-IgE as a mast cell-stabilizing therapeutic agent

After nearly 20 years of development, humanized monoclonal anti-IgE antibodies have been shown in about 20 phase II and III clinical trials to be effective and safe in treating allergic asthma, perennial and seasonal allergic rhinitis, and allergic reactions to peanuts. Omalizumab has been approved in the United States, European Union, and several other countries for treating patients 12 years and older with moderate-to-severe allergic asthma. Although anti-IgE is often referred to as an IgE-neutralizing antibody and can block IgE's binding to high-affinity IgE Fc receptors (FcepsilonRI) on mast cells and basophils, it has multiple immunoregulatory effects. One such effect is that as the result of depleting free IgE, FcepsilonRI on mast cells and basophils is downregulated to less than 5% within a few weeks to a few months of anti-IgE treatment. This renders the mediator-packed inflammatory cells insensitive to allergen stimulation. Hence this therapeutic anti-IgE represents a new class of mast cell-stabilizing agents, reducing FcepsilonRI density on mast cells and basophils and causing them to be insensitive to allergens. This mechanism contrasts with that of cromones, mast cell-stabilizing agents that retard Ca++ mobilization and the degranulation process, thus deflating the intracellular activation signal triggered by IgE-FcepsilonRI aggregation.     

Human basophils as effectors and immunomodulators of allergic inflammation and innate immunity

Abstract Basophils have often stood in the shadow of their tissue–fixed mast cell counterparts which share some, common features, such as high–affinity IgE receptor expression and the ability to release histamine. That rodent mast cells produce a variety of pro–allergic and inflammatory cytokines has further added to the deception that basophils only play a minor role in allergic inflammation. Surprisingly, in humans, basophils, but not mast cells, appear to be the prime early producers of the Th2–type cytokines IL–4 and IL–13, which perform several crucial functions in initiating and maintaining allergic responses. This putative immunomodulatory role of basophils is supported further by their ability to express CD40 ligand, which, together with IL–4 and IL–13, serve as inductors of B–cell proliferation and class switching to IgE and IgG4. Moreover, human basophils are the main cellular source for rapid IL–4 generation, a mandatory requirement for the development of Th2 responses. Recent specific staining techniques have localised basophils in various tissues affected by allergic diseases and it appears likely, but remains to be proven, that the interaction of basophils, T cells and B cells at these sites propagate pro–allergic immune responses. Additionally, basophil activation is not restricted to antigen–specific IgE crosslinking but can be caused in non–sensitised individuals by parasitic antigens, plant lectins and viral superantigens binding to non–specific IgEs. Finally, the presence of novel IgE–independent receptor targets that cause trafficking and Th2 cytokine release from basophils further underlines their potential role in innate as well as adaptive immunity.     

Passive IgE-sensitization by blood transfusion

BACKGROUND: To study the mechanisms of passive sensitization of patients receiving plasma containing IgE antibodies to a defined allergen. METHODS: When required for medical reasons, regular donor plasma with IgE antibodies to timothy grass allergen (8-205 kU(A)/l), was given. Kinetics of IgE antibodies in the recipients' serum and his/her basophil allergen threshold sensitivity, CD-sens, was monitored up to 2-3 weeks after transfusion. The IgE antibodies were quantitated by ImmunoCAP. The CD-sens in plasma recipients, determined by CD63 up-regulation, was measured by flow cytometry and compared to CD-sens of patients with allergic asthma and/or rhinitis. RESULTS: There was a significant correlation (r = 0.98; P < 0.001) between amount of IgE antibody given and recipient serum peak concentration. The T(1/2) for IgE antibody in circulation was 1.13 days (95% confidence limit 0.35-1.91 days). The recipients became CD-sens positive already 3 h after transfusion. The CD-sens peak was observed after 3.4 days and the value were correlated (r = 0.68; P < 0.02) to the amount of IgE antibody transfused and were of the same magnitude as found in allergic patients. The T(1/2) of CD-sens indicated two populations of basophils; one with a CD-sens decrease T(1/2) of 4 days and one of 10 days. CONCLUSION: Transfused IgE antibodies will sensitize mast cells and basophils to CD-sens levels similar to those of allergic patients. The recipients expressed 'slow' or 'rapid' CD-sens decline, indicating two different basophil populations. After transfusion of plasma with >10 kU(A)/l IgE antibody the recipient could have allergen reactive basophils for up to 7 weeks.     

The anti-inflammatory effects of omalizumab confirm the central role of IgE in allergic inflammation

Anti-IgE therapy with omalizumab reduces serum levels of free IgE and downregulates expression of IgE receptors (Fc epsilonRI) on mast cells and basophils. In the airways of patients with mild allergic asthma, omalizumab reduces Fc epsilonRI+ and IgE+ cells and causes a profound reduction in tissue eosinophilia, together with reductions in submucosal T-cell and B-cell numbers. In patients with seasonal allergic rhinitis, omalizumab inhibits the allergen-induced seasonal increases in circulating and tissue eosinophils. Omalizumab decreases Fc epsilonRI expression on circulating dendritic cells, which might lead to a reduction in allergen presentation, T(H)2 cell activation, and proliferation. As a systemic anti-IgE agent, omalizumab has demonstrated clinical efficacy in patients with moderate and severe allergic asthma and in those with seasonal and perennial allergic rhinitis, as well as in patients with concomitant allergic asthma and allergic rhinitis. The anti-inflammatory effects of omalizumab at different sites of allergic inflammation and the clinical benefits of anti-IgE therapy in patients with allergic asthma and allergic rhinitis emphasize the fundamental importance of IgE in allergic inflammation.     

Profound reduction in allergen sensitivity following treatment with a novel allergy vaccine

A novel approach is described for the treatment of IgE-mediated allergic reactions which is based on the induction of a strong anti-IgE response in the host. Vaccination of ovalbumin-sensitized rats with constant domains two and three of rat IgE coupled to a heterologous carrier protein resulted in a profound decrease in serum levels of IgE, and later in a nearly complete block of histamine release from mast cells and basophils upon challenge with either a cross-linking polyclonal anti-IgE antiserum or a specific allergen.     

Important and specific role for basophils in acute allergic reactions

IgE‐mediated allergic reactions involve the activation of effector cells, predominantly through the high‐affinity IgE receptor (FcεRI) on mast cells and basophils. Although the mast cell is considered the major effector cell during acute allergic reactions, more recent studies indicate a potentially important and specific role for basophils and their migration which occurs rapidly upon allergen challenge in humans undergoing anaphylaxis. We review the evidence for a role of basophils in contributing to clinical symptoms of anaphylaxis and discuss the possibility that basophil trafficking during anaphylaxis might be a pathogenic (to target organs) or protective (preventing degranulation in circulation) response. Finally, we examine the potential role of basophils in asthma exacerbations. Understanding the factors that regulate basophil trafficking and activation might lead to new diagnostic and therapeutic strategies in anaphylaxis and asthma.     

The basophil-specific ectoenzyme E-NPP3 (CD203c) as a marker for cell activation and allergy diagnosis

Basophils are effector cells of allergic reactions. These cells produce and store a number of vasoactive and immunomodulatory mediators. During an allergic reaction, basophils can release their mediator substances into the extracellular space and thus contribute to the clinical picture and symptoms in allergy. The phenotypic hallmark of basophils is expression of high-affinity IgE receptors (Fc epsilon RI) on their cell surface together with expression of the activation-linked molecule CD203c. This ectoenzyme is located both on the plasma membrane and in the cytoplasmic compartment of basophils. Cross-linking of the Fc epsilon RI by an allergen or anti-IgE antibody results in a rapid upregulation of intracellular CD203c molecules to the cell surface and is accompanied by mediator release. CD203c is therefore a promising target molecule for a flow cytometry-based test to analyze sensitized individuals and patients with type I allergy. In the present article, we review the current knowledge of CD203c with special regard to its tissue distribution and regulation in basophil activation. In addition, we discuss the application of CD203c in allergy diagnosis.     

Differences in response to anti-IgE and to anti-IgG in basophils from patients with bronchial asthma

Peripheral blood basophils of thirty-eight patients with bronchial asthma were examined for their reactivity to anti-IgE and anti-IgG antisera. Basophils of patients with serum IgE levels of more than 1001 i.u./ml reacted strongly to anti-IgE. Basophils of patients with serum IgE levels of less than 100 i.u./ml had a tendency to react more strongly to anti-IgG. An index (basophil ratio) was devised to compare the patient basophil reactivity to anti-IgE and anti-IgG. This basophil ratio was lower (IgE dominant) in the atopic cases which usually exhibited a high serum IgE level. Most cases with low serum IgE exhibited a high basophil ratio (IgG dominant). The basophils of seven intractable patients reacted more strongly to anti-IgG than to anti-IgE regardless of the serum IgE level.     

Anti-immunoglobulin E treatment with omalizumab in allergic diseases: an update on anti-inflammatory activity and clinical efficacy

Omalizumab is a humanized monoclonal anti-IgE antibody developed for the treatment of allergic disease, with established efficacy in patients with moderate-to-severe allergic asthma and in patients with intermittent (seasonal) and persistent (perennial) allergic rhinitis (AR). Omalizumab is known to result in a marked reduction in serum levels of free IgE and down-regulation of IgE receptors on circulating basophils. Recent work has shed further light on its mechanism of action, showing significant and profound reductions in tissue (nasal and bronchial) eosinophils and in bronchial IgE+ cells (mast cells), as well as T cells and B cells. Omalizumab treatment was also shown to be associated with down-regulation of IgE receptors on circulating (precursor) dendritic cells, suggesting that blocking IgE may inhibit more chronic aspects of allergic inflammation involving T cell activation. Further work with omalizumab demonstrated it to have important benefits in patients with poorly controlled asthma despite high-dose inhaled corticosteroid therapy, and analysis of clinical data suggests that the patients who are the best 'responders' to anti-IgE treatment are those with asthma at the more severe end of the spectrum. Notably, systemic anti-IgE therapy with omalizumab has been shown to improve symptoms, quality of life and disease control (asthma exacerbations) in patients with concomitant asthma and persistent AR. These impressive clinical data and the studies elucidating the anti-inflammatory profile of omalizumab also serve to emphasize the fundamental importance of IgE in the pathogenesis of allergic diseases.     

Anti-IgE therapy

IgE has a central role in the initiation of allergic hypersensitivity reactions. Pioneered by the development of humanized monoclonal antibodies directed against IgE, anti-IgE therapy represents an original approach to the treatment of allergic disease and has demonstrated promise in preventing the symptoms of asthma, allergic rhinitis, and food allergy. The primary mechanism of action of anti-IgE therapy is the reduction in serum levels of IgE and FcepsilonRI expression on mast cells and basophils.     

Renaissance of the blocking antibody concept in type I allergy

Formation of IgE antibodies against per se harmless antigens (i.e. allergens) is the hallmark and key pathomechanism of type I allergy, a hypersensitivity disease affecting more than 25% of the population. Classical experiments performed more than 65 years ago demonstrated that allergen-specific IgG antibodies, termed blocking antibodies, can antagonize the cascade of allergic inflammation resulting from allergen recognition by IgE antibodies. However, controversial results have questioned the protective role of IgG antibodies in allergic diseases. Here, we review recent data demonstrating that blocking antibodies inhibit allergen-induced release of inflammatory mediators from basophils and mast cells as well as IgE-facilitated allergen presentation to T cells, thus leading to suppression of T cell activation. Furthermore, it has been reported that the development of blocking antibodies is associated with reduced boosts of allergen-specific IgE production in patients receiving allergen-specific immunotherapy. These findings suggest that blocking antibodies have protective activity by inhibiting immediate as well as late inflammatory responses and long-term ameliorating activity on the allergic immune response by antagonizing the underlying IgE production. Induction of blocking antibodies is thus an important mechanism underlying allergen-specific immunotherapy. In addition, passive administration of blocking antibodies may be considered as a potential therapeutic strategy for allergic diseases.     

Appearance of basophilic cells/mast cells and IgE antibodies in liquid human cell cultures

Maturation of basophils/mast cells as well as formation of IgE antibodies are parts of the immune response to antigens. We have analyzed these important constituents of the allergic inflammatory response in vitro, cultivating human peripheral blood cells from allergic and normal individuals. Mesenteric lymph nodes were also cultured from two normals. In liquid cultures of peripheral blood cells, the number of small basophils initially present decreased and large basophilic cells appeared over a 2-week period. In normals, the maturation of large basophils from precursor cells could be stimulated with supernatants from peripheral blood cell cultures from atopic individuals stimulated with the relevant allergen. The IgE levels declined in cultures of peripheral blood, whereas the IgE antibodies in cultures of cells from a mesenteric lymph node from one of the nonallergic individuals increased during the 2-week period. In separate cultures, the IgE formation could not be reproducibly stimulated by antigen or mitogen. The study shows the possibility to stimulate the maturation of basophil/mast cell precursor cells present in the blood of allergic and healthy individuals with antigen-induced factors. In contrast, the IgE formation by blood cells was difficult to stimulate.