Increased expression levels of CD300c on basophils from allergic individuals☆

Immunoglobulin E (IgE)-mediated allergy refers to the adverse reaction in some patients caused by the crosslinking of the high-affinity IgE receptor (FcεRI) on basophils and mast cells by allergenspecific IgEs. Once the contact with allergens is established, immunomodulatory therapeutic agents to prevent the onset of allergic symptoms are currently quite limited. Blocking the interaction of specific IgE with the FcεRI that is present in basophils and mast cells is an important target in antiallergic therapy. Clinical responses have been observed after the administration of omalizumab, a humanized anti-IgE antibody, to patients with food allergy, which correlated with the suppression of degranulation of basophils. These results indicate that actions aimed to blocking or modulating the IgE/FcεRI axis represent a promising strategy for the treatment of allergy and anaphylaxis. Therefore, in order to develop new immunomodulatory therapies, it is very important to characterize cell surface receptors capable of modulating IgE-mediated activation threshold in basophils and mast cells.

Immunoglobulin E (IgE)-mediated allergy refers to the adverse reaction in some patients caused by the crosslinking of the high-affinity IgE receptor (FcεRI) on basophils and mast cells by allergenspecific IgEs. Once the contact with allergens is established, immunomodulatory therapeutic agents to prevent the onset of allergic symptoms are currently quite limited. Blocking the interaction of specific IgE with the FcεRI that is present in basophils and mast cells is an important target in antiallergic therapy. Clinical responses have been observed after the administration of omalizumab, a humanized anti-IgE antibody, to patients with food allergy, which correlated with the suppression of degranulation of basophils. 1,2 These results indicate that actions aimed to blocking or modulating the IgE/FcεRI axis represent a promising strategy for the treatment of allergy and anaphylaxis. Therefore, in order to develop new immunomodulatory therapies, it is very important to characterize cell surface receptors capable of modulating IgE-mediated activation threshold in basophils and mast cells. 3 The human CD300 family of receptors consists of 8 receptors expressed in both myeloid and lymphoid lineages. 4 It has been recently demonstrated that the phosphatidylserine and phosphatidylethanolamine binding CD300c receptor acts as a co-stimulatory molecule during basophil activation through the IgE/FcεRI axis. CD300c cross-linking significantly augmented IgEmediated basophils degranulation and cytokine production in a process involving increased calcium mobilization and phosphorylation of signaling intermediates such as protein tyrosine kinase (Syk) and extracellular signal-regulated kinases (ERK). Moreover, it was observed that basal expression levels of CD300c on basophils from IgE-dependent cow's milk allergic children are higher than those from healthy control children, suggesting that CD300c could be used as a biomarker in the diagnosis of the IgE-dependent allergic pathology. 5 In order to further assess the clinical relevance of those findings, we have studied the expression of CD300c on basophils from patients with two IgEdependent allergies. Clinical features from patients are shown in Table 1. We collected peripheral blood samples from 3 different cohorts: 1) adults with dust mites allergy (n ¼ 36), 2) adults with grass pollen allergy (n ¼ 22), and 3) non-allergic control individuals (n ¼ 26).
To identify basophils among the peripheral blood mononuclear cells (PBMCs), we have used a staining strategy based on the expression of the surface receptor CD123 and the absence of human leukocyte antigen -DR isotype (HLA-DR) (shown in the article's online supplementary Figure S1). First, we analyzed the expression of CD63, a basophil activation marker which is rapidly mobilized to the cell surface by polyclonal anti-IgE and allergens, as well as other degranulation stimuli. 6 In agreement with previous publications, 7 we observed that subjects with dust mites and/or grass pollen allergy show a significant increase in the median fluorescence intensity (MFI) of CD63 compared with basophils from non-allergic subjects, indicating that they are activated in vivo ( Fig. 1A and online supplementary Figure S2). We cannot confirm that the increased expression on CD63 is exclusively due to a higher expression on basophils, and that other possibilities, as for example a greater adhesion of platelets, have Next, we analyzed cell surface expression levels of the inhibitory and activating CD300a and CD300c receptors, respectively. We observed that surface CD300c expression levels are significantly higher in basophils from individuals with an IgEdependent allergy (dust mites and grass pollen) than in those from non-allergic individuals (Fig. 1B). This increased expression of CD300c could diminish basophils FcεRI-mediated activation threshold. 5 It has been previously described that the basal expression of the CD300a inhibitory receptor is lower in basophils from patients with birch pollen allergy than in basophils from healthy control subjects. 11 However, we have not seen significant differences between healthy and allergic individuals related to CD300a expression (Fig. 1C), which is in agreement with previous results in cow's milk allergic children. 5 We performed correlation analysis between the intensity of CD300c and CD63 expression, but we did not find any significant result (data not shown). As it has been mentioned before, an association between the severity of the hypersensitivity symptoms and the levels of CD300c expression on basophils in cow's milk allergic children has been described. 5 However, in this study the vast majority of the recruited patients (88%) were classified to have mild symptomatology (Table 1), and therefore it was not adequate to perform this analysis.
Unlike mite allergy, grass pollen allergy is a seasonal affection, and the exposure of patients to the allergen varies throughout the year. Although the great majority of samples have been collected during the grass season, we have two samples of the same individual collected at different times of the year, one in grass season and the other not. We observed that the expression of CD300c is higher during the grass pollen season than out of the season (MFI 492 vs 376, respectively). This may suggest that the exposure of allergic patients to higher amounts of allergen could induce an up-regulation of basophil activating receptors such as CD300c. The specific mechanism regulating CD300c  CD300c (B) and CD300a (C) on basophils from non-allergic (blue), allergic to dust mites (red) and allergic to grass pollen (green) individuals. Each dot represents a donor, means AE SEMs are shown. *P < .05, **P < .01, ****P < .0001 expression in these allergic patients is still unknown and deserves further studies.
To date, the only tested stimulus able to upregulate CD300c expression is IL-3. 5 This is a very important cytokine for the development, maturation, and survival of basophils. 12 This cytokine is known to markedly increase the activation and release of mediators from basophils in IgE-dependent responses, 12 and the autocrine priming with IL-3 has been described as an important mechanism behind the hyperreactive nature of basophils in the allergic disease. 13 We analyzed IL-3 in plasma from allergic subjects, but the levels of this cytokine were mostly undetectable. It is possible that the mild symptoms exhibited by the majority of patients may be related to the observed results. Based on our data, we propose that baseline expression levels of CD300c, together with CD63 expression, on human basophils could be helpful for the diagnosis of IgE-dependent allergies. Furthermore, considering that CD300c is capable of modulating the threshold of IgE-mediated activation in human basophils, 5 we believe that, as previously demonstrated, 5 an increased expression of CD300c decreases the IgE-dependent activation threshold of basophils in allergic individuals.

Conflict of interests
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.