Kinetics of basophil suppression
Studies describing the kinetics of basophil suppression have provided some of the first insights into the mechanisms of that suppression. In 1996, Jutel et al. demonstrated that histamine release from antigen-stimulated peripheral mononuclear cells was decreased in bee-allergic patients after undergoing the build-up phase of ultra-RIT , suggesting that the early tolerance induced by the immunotherapy resulted in basophil suppression. Since then, the onset of basophil suppression during immunotherapy has been studied in clinical trials with a variety of immunotherapy schedules and routes.
In one peanut oral immunotherapy trial (OIT), during which dose escalation occurred over months, the onset of basophil suppression in immunotherapy-treated patients occurred during the first 4 months of therapy compared to baseline values before initiation of OIT, and persisted through the immunotherapy period . Similarly, Ebo et al. noted that in patients treated with RIT for Vespula vulgaris hypersensitivity noted that basophil CD63 up-regulation was not significantly different from baseline at 5 days, but was significantly decreased at 6 months .
Another study of RIT in 48 patients with Hymenoptera venom hypersensitivity did not find any change of in vivo basophil CD63 expression between the pre- and post-build-up phase; however, in 20 of those patients who were examined 1 week after completion of RIT, there was a significant decline in CD63 basophil expression . An important methodological difference of this study is the measurement of in vivo activation, which may influence the ability to detect differences in basophil activation.
Interestingly, Mikkelsen et al. studied serial basophil activation in patients undergoing a mix of cluster and traditional subcutaneous immunotherapy to Vespula vulgaris, with a 7–11-week build-up phase. Suppression of in vitro antigen-stimulated basophil activation was seen at 3 weeks and returned subsequently to the initial baseline, where it remained at weeks 7 and at the time of maintenance initiation . As clinical outcomes of immunotherapy were not reported, the absence of sustained basophil suppression could be attributed to a lack of clinical efficacy.
Comparison of the kinetics of basophil suppression with immunotherapy is hampered by the need for serial measurements through the build-up phases of immunotherapy, as basophil suppression may be an early phenomenon during the course of immunotherapy. This may be particularly true if the immunotherapy protocol involves daily exposure to antigen, which may be anergy-inducing, versus intermittent allergen dosing, which may not have this effect at all.
Extrinsic changes during immunotherapy
The serological changes that occur during immunotherapy are likely a primary mechanism affecting basophil and other effector cell activation. As degranulation is dependent on antigen-stimulated specific IgE cross-linking on the surface of basophils, modulation of basophil activation has been speculated to correlate with levels of specific IgE. Evidence for early transient increase in specific IgE has been seen in oral and sublingual immunotherapy [16,17,30] with subsequent decrease in specific IgE after 1 or more years of immunotherapy [15–17], although some studies have not seen significant change [20,29,35].
Factors that influence IgE-mediated basophil activation (and therefore may also impact the suppression of basophil activation) include surface density of the high-affinity IgE receptor (FcεRI), fraction of membrane-bound-specific IgE (which is influenced by the ratio of specific to total IgE in the serum) , the clonality of the antigen-specific IgE, biochemical properties of the allergen and intrinsic basophil sensitivity . For instance, an elegant set of studies devised by Christensen et al. utilized recombinant specific IgE with predetermined affinity to Der p 2 to demonstrate the effect of clonality of IgE on basophil activation . This paper demonstrates that both the affinity and composition of the surface allergen-specific IgE impacts basophil degranulation. The inconsistent changes in specific IgE levels associated with clinically effective immunotherapy suggest that other immunotherapy-induced changes are mechanistically more important.
An early and sustained increase in allergen-specific IgG4 has been detected more reproducibly [16,17,20,28,29,34,38–40], although older studies did not find an association between IgG and clinical improvement [41,42]. Direct suppression of basophil activation by allergen-specific IgG4 could occur by either blocking IgE-allergen binding and/or signalling via inhibitory IgG receptors. Several studies have shown that IgG4-containing serum from patients post-immunotherapy can suppress basophil activation [18,35,40,43,44] or decrease of β-hexosaminidase release from rat basophilic leukaemia (RBL) cells .
One model of blocking IgE-allergen interaction with IgG generated recombinant IgG with Phl p 2 epitope specificity from a human grass-allergic donor's IgE , to demonstrate in vitro inhibition of IgE-grass pollen complex binding to CD23 of B cells as well as decreased histamine release from antigen-stimulated basophils. Subsequent studies of both subcutaneous and sublingual grass-pollen immunotherapy have shown the induction of allergen-specific IgG antibodies with IgE-allergen blocking capability and their persistence even after cessation of immunotherapy [46,47].
Using another mechanistic approach, Uermosi and colleagues devised a chimeric Fel d 1 IgG antibody to demonstrate decreased degranulation of basophils from patients with cat allergies . Suppression was effective with either IgG1 or IgG4 and was increased further with the use of two or three different epitope specificities of the IgG antibodies. This increased suppression was speculated to be due to more efficient FcγRIIB cross-linking. Interestingly, previous work suggests that IgG epitope specificity is affected by immunotherapy and increases suppressive activity after immunotherapy .
IgG-mediated basophil suppression includes signalling through low-affinity IgG receptors (FcγIIRA and FcγIIRB), which are expressed on the surface of circulating basophils. Stimulation of these receptors can induce inhibitory signalling through their ITIMs. Using cat-specific IgG from serum of cat allergic patients on subcutaneous immunotherapy, Cady et al. demonstrated that suppression of CD203c expression on basophils acts via inhibitory receptors FcγIIRA and FcγIIRB . Moreover, co-stimulation of FcεRI and FcγIIR on basophils results in suppression of basophil activation and increase in SHP-1 levels . Another study utilized a chimeric IgG antibody to bind both Fcε and Fcγ receptors on basophils, which decreased antigen-specific basophil degranulation from atopic donors . Furthermore, a chimeric fusion protein of Fcγ-Fcε that bound both FcγIIR and FcεRI was found to decrease human basophil activation as well as Syk phosphorylation in vitro. This type of inhibitory mechanism has been used in an antigen-specific manner by Zhu et al., who devised a chimeric fusion of Fcγ to cat allergen Fel d 1 designed to aggregate FcγRIIB and FcERI to demonstrate a decrease in histamine release from basophils of cat-allergic patients .
The above studies suggest that extrinsic factors, such as IgG4 and FcgRII stimulation, may contribute to the suppression of basophil activation during immunotherapy.
Intrinsic basophil changes during suppression
As noted above, suppression of basophil activity with immunotherapy has been evidenced by suppression of markers of basophil activation. IgE-dependent basophil activation begins with cross-linking of antigen-specific surface IgE, with subsequent recruitment and phosphorylation of tyrosine kinases Lyn and Syk, leading to activation of Phosphoinositide 3-kinase (PI3K) and phospholipase C activation. Intracellular calcium mobilization from inositol triphosphate (IP3) generation leads to secretion and/or de novo synthesis of basophil allergic mediators, including histamine, cytokines and leukotrienes.
Variability in human basophil mediator release has been linked to levels of these intracellular signalling molecules. About 10–20% of the human population have ‘non-releaser basophils’, which do not secrete histamine to anti-IgE stimulation . Basophil histamine release has been correlated with expression levels of Syk and phosphatidylinositol 5′ phosphatase (SHIP) expression in the human population . A composite characteristic of basophil activation can be summarized using the term ‘intrinsic basophil sensitivity’ to refer to the intracellular signalling characteristics of patients' basophils.
Additional studies on pathways of signal termination of antigen-stimulated IgE-FcεRI signalling pathways substantiated the role of syk and actin-mediated pathways . However, these studies also suggest that the pathways of signal self-termination may not be the same as those of anergy, or desensitization, in basophils. Several approaches to study basophil anergy or desensitization have been employed in vivo. The use of suboptimal antigen stimulation, which would not stimulate maximal mediator release from basophils for longer periods of time (24 h), resulted in reduced Syk but not Lyn levels . Another approach was to stimulate basophils in vitro in calcium-free conditions, which would inhibit mediator release. When basophils were stimulated in a calcium-free environment in the presence of actin inhibitors mediator release was unchanged, suggesting that although actin-mediated pathways may play a role in antigen-IgE-FcεRI signal termination, they do not impact basophil anergy .
The in vitro induction of basophil desensitization most similar to the clinical model of immunotherapy uses repeated antigen stimulation, which was performed by Lund et al. to demonstrate grass-specific desensitization of basophils from grass allergic donors . A similar model of antigen-induced anergy suggested that Syk and PI3 are not involved in mechanisms of anergy . A study inducing desensitization by using increasing antigen concentrations to stimulate antigen-specific IgE-sensitized bone-marrow-derived mast cells suggested the internalization of FcεRI–IgE-antigen complexes is impaired during the process. This is contrary to previous speculations about possible FcεRI internalization as a mechanism for decreasing antigen sensitivity of allergen effector cells . Whether this applies to human basophils remains to be studied, as FcεRI endocytosis has been reported in these basophils . Hence, further studies on intrinsic changes in basophil activation during immunotherapy are needed and may highlight potential avenues for therapeutic intervention.