Spontaneous basophil responses in food-allergic children are transferable by plasma and are IgE-dependent.

To the Editor: Early studies showed that up to 80% of children with food allergy demonstrate high spontaneous basophil histamine release (SBHR).1 An association with IgE was suggested because basophils from food-allergic (FA) children additionally released histamine to the IgE-dependent histamine-releasing factor.2 However, the exact mechanism(s) responsible for SBHR, including the dependency for IgE, remains poorly understood. We recently reported that SBHR significantly decreased in children undergoing sublingual immunotherapy/oral immunotherapy (SLIT/OIT) for cow's milk allergy,3 prompting further investigations of this response and other markers of basophil hyperresponsiveness. Basophils were isolated from the blood of 38 FA (all to cow's milk) and 18 nonallergic children, as described in the Methods section of this article's Online Repository available at www.jacionline.org. Twenty-four children had been followed at baseline and during the SLIT/OIT protocol.3 Thus, baseline values for several basophil parameters investigated in this subgroup are restated herein to demonstrate correlative associations not previously reported. Supporting demographic and clinical information regarding all subjects is summarized in Table E1 of this article's Online Repository available at www.jacionline.org. Diagnosis of milk allergy was based on a convincing history of acute reactivity to cow's milk (rxn+) and a milk-specific IgE level of more than 0.10 KUA/L (cap+). All FA subjects were strictly avoiding milk at the time blood was drawn. Control subjects had no history of acute reaction to any food (rxn−), had never avoided any foods, and were currently tolerating milk in their diet. Although, 3 of the 18 control subjects did have low levels of milk-specific IgE (1.52 KUA/L being the highest), these were less than those in the FA group (median, 52.5 KUA/L; range, 1.9-1108.0 KUA/L). Basophils from 13 FA and 10 control subjects were investigated for histamine and IL-4 responses as shown in Fig 1, A. Eleven of the 13 FA subjects demonstrated SBHR (17%-48%, median of 22%), while this phenotype was not evident in the 10 control subjects, including those testing IgE-positive to milk but having no reaction history (Con−cap+/rxn−). Basophil histamine release (BHR) to milk occurred among the FA subjects but was absent among the controls. All subjects demonstrated BHR to anti-IgE and calcium ionophore (positive controls). Basophils from FA subjects additionally secreted IL-4 spontaneously, with response patterns that mirrored those for histamine. SBHR and spontaneous IL-4 significantly correlated (Fig 1, B; r = 0.66, P = .01), yet neither correlated with total or milk-specific IgE levels (data not shown). FIG 1 A, SBHR and IL-4 (spont.), compared with induced, among FA and control (Con) subjects (Cap+-milk-specific IgE; rxn+-clinical reactivity to milk; anti-IgE—an activating anti-IgE; {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}} ... We previously found that constitutive expression of CD203c and SBHR decreased in subjects who had undergone SLIT/OIT.3 This prompted our analysis and subsequent discovery that SBHR significantly correlated (although modestly) with constitutive expression of CD203c, but not with CD63 (Fig 1, C). In addition, protein levels of spleen tyrosine kinase (syk)—a marker of IgE-dependent activation,4 showed a similar correlation with SBHR (Fig 1, D) and were approximately 2-fold greater in basophils of FA subjects than in basophils of controls (Fig 1, E). As expected, syk correlated with BHR to milk extract, but not to release induced by f-met—an IgE-independent stimulus (Fig 1, F and G). We next asked whether SBHR was a property intrinsic to allergic basophils, or whether this phenotype could be passively transferred to basophils from nonallergic subjects. Remarkably, several markers of basophil hyperresponsiveness were exhibited by normal basophils following passive sensitization using plasma from FA subjects, but not with control plasma spiked with matching amounts (500 ng/mL) of myeloma IgE. Respective values for SBHR (35% ± 8% vs 2.5% ± 0.5% total histamine) and IL-4 (45 ± 5 vs 6 ± 2 pg/106 basophil) are shown in Fig 2, A. Transfer of both responses was ablated by neutralizing IgE with omalizumab during the passive sensitization step whereas neutralizing IL-3 activity—a potent enhancer of basophil responses—did not. As expected, reactivity to milk was likewise transferred to nonallergic basophils and prevented by neutralizing IgE. Responsiveness to goat anti-human IgE antibody was not inhibited by omalizumab, indicating the retention of residual receptor-bound IgE despite lactic acid treatment. FIG 2 Markers of hyperresponsiveness are transferred to normal basophils via plasma from FA (milk pt) vs control (NA) subjects and are inhibited by omalizumab. A, Values (mean ± SEM) for BHR (n = 3) and IL-4 secretion (n = 2). #P < .05 (vs control). ... We further found that CD203c and CD63 expression on nonallergic basophils spontaneously increased after passively sensitizing with plasma from FA subjects, but not with plasma from nonallergic control subjects (Fig 2, B). Reactivity to milk and anti-IgE was also transferred as expected. This phenomenon was IgE-dependent because the addition of omalizumab during passive sensitization inhibited both the spontaneous induction of CD203 (and CD63—data not shown) and the reactivity to milk (Fig 2, C). In conclusion, we confirm herein the findings of May,1 who showed high SBHR among FA subjects, and further demonstrate that this phenotype correlates with spontaneous IL-4 secretion, higher constitutive expression of CD203c, and increased levels of intracellular syk. Most significantly, we demonstrate that SBHR and IL-4 secretion, along with increased CD203c expression, are all transferable to basophils of nonallergic subjects via passive sensitization using plasma from FA individuals. Transfer of these markers is completely suppressed by omalizumab, pointing to the requirement of IgE (an extrinsic factor) for these responses. However, SBHR did not correlate with total or milk-specific IgE (data not shown), reflecting evidence that intracellular syk levels are rate limiting in determining basophil sensitivity to IgE-dependent stimulation, not the overall amount of IgE.4 Whether the transfer of SBHR (or other markers) has diagnostic value by predicting whether one is at risk for adverse reactions to milk (or other hypersensitivities) remains to be evaluated, but recent studies suggest that this may be the case. For instance, higher expression of CD203c has been associated with asthma exacerbations,5 and was greater among patients with more severe clinical reactivity to milk.6 OIT and anti-IgE therapy have been shown to reduce CD203c, suggesting that therapeutic interventions can modulate its expression.3,7 IgE/allergen complexes have been implicated in SBHR,8 even though in vitro responses (often exceeding 50% of total) seem incompatible with survival if the same intensity was to be achieved in vivo. In addition, higher expression of CD203c by basophils from allergic subjects may indicate a subthreshold level of activation (or priming), perhaps reflecting histamine release in a manner (eg, piecemeal) distinct from anaphylactic degranulation.9 We hypothesize that an unknown secondary mechanism(s) in vivo prevents full-blown anaphylactic degranulation of basophils, possibly explaining, in part, why such reactions are not more common. This suppressive activity seems lost once the cells are processed ex vivo and cultured, thus allowing subthreshold activation to proceed to full-blown degranulation, with induction of CD63 and SBHR. If so, the exact mechanism(s) preventing this progression in vivo requires further investigation. The findings presented herein could help provide the momentum, and newfound tools, for pursuing this hypothesis.