Involvement of functional autoantibodies against the high-affinity IgE receptor (FcɛRI) of dermal mast cells and basophils has been largely documented (2, 5–7) and the term of AICU now identifies a subset of patients in which these autoantibodies are the cause of the lesions, as suggested by a positive in vivo response to intradermal injection of autologous serum (3, 4). Although numerous attempts have been made to devise specific and sensitive in vitro tests for AICU, including enzyme-linked immunosorbent assay and other immunobinding assays (6, 7, 11), the current gold standard laboratory test is the demonstration and measurement of histamine release from target basophils or dermal mast cells (4, 9). It is well known that basophils activation can be detected by flow cytometric measurement of CD63, a tetraspan granule protein that appears de novo on basophils membrane after degranulation (14); in resting basophils, CD63 is anchored in basophilic intracytoplasmic granules and, as a consequence of cell degranulation, it is expressed with a high density on activated basophils and mirrors mediators release (15–17). Two previous studies by Wedi et al. (18) and Gyimesi et al. (19) based on different laboratory approach demonstrated that sera of a subset of CU patients with positive ASST are able to induce CD63 expression on atopic donors basophil when identified with a two-colour flow cytometric method using anti-IgE and anti-CD63 MoAbs. On the basis of these interesting data, the goal of our study was to standardize the CU serum-induced CD63 expression assay on a large series of patients by means of a new tricolour flow cytometric method which allows to accurately quantify activated CD63+ cells on a selected population of CD123+ HLA-DR- basophils from whole blood, without IL-3 pretreatment (22, 23). Applying this method for the first time in a large series of CU patients, our results demonstrated that (i) sera from ASST+ CU patients are able to induce CD63 expression on donors basophil in a significant higher manner when compared with ASST− CU patients and healthy donors sera, (ii) ASST+ CU serum activity on CD63 expression was significantly enhanced when basophils from an atopic sensitized donor were used, (iii) according to the ROC-generated cut-off value of 15%, the sensitivity and specificity of the serum-induced CD63 expression assay was 95.5% and 90.5% respectively, (iv) ASST+ CU serum induced CD63+ basophils from DA are significantly decreased below the cut-off during CyA treatment, in parallel with the complete clearing of skin lesions and the reduced ASST response. Wedi et al. (18), using whole blood primed with IL-3, demonstrated serum-induced CD63 expression in 70% of ASST+ CU patients, as well as in 45% of ASST− CU and in 35% of control subjects (both atopic and nonatopic), thus concluding that CU serum activity inducing CD63 expression cannot be regarded as a diagnostic marker of CU. Gyimesi et al. proposed a modified protocol of CU serum-induced CD63 expression assay using dextran-sedimented washed leucocytes from highly sensitized atopic donors, thus avoiding preliminary priming with IL-3. With this method they reported higher values of sensitivity and specificity in respect to Wedi et al., documenting CD63 expression in 91% of ASST+ CU patients and in 17% of ASST− CU patients, with no induction of CD63 in both healthy and disease control groups (19). On one hand, our results confirm, in agreement with Gyimesi et al., that ASST+ CU sera are able to significantly induce CD63 expression on basophils, in contrast to ASST− CU and healthy donors sera. On the other hand, using a model consisting of whole blood without IL-3 pretreatment and a different strategy of basophils identification, we statistically found values of sensitivity and specificity of the serum-induced CD63 assay higher than those observed in the previous studies. We believe that the improved diagnostic performance may be due especially to the new tricolour flow cytometric method used in our study which allows an high basophil recovery even in whole blood. Indeed, the identification of basophils using prior protocols relied on a single IgE-labelling, although it is known that FcɛRI expression can vary considerably on cell surfaces from one subject to another (24). CD123, costitutively expressed at high density on basophils, is known to be a useful marker for basophil identification, as its expression is less variable than surface IgE and is independent of the allergy status of the donor (20, 23). With a double gating strategy of basophil phenotyping which isolate low side scatter, CD123+ HLA-DR- cells, the expression of CD63 can be easily and accurately quantify in this selected population. The higher sensitivity of this method could also explain the ASST+ CU serum activity in inducing CD63 expression also on DNA basophils, although the use of DA whole blood is highly recommended to obtain a better results in terms of the magnitude of the serum challenge.
Taking in mind the crucial need for inter-laboratory standardization in clinical decision-making, we suggest that the easy recognition of basophils in whole blood and the reliable assessment of their activation by means of this tricolour FCM protocol could be the most useful tool for in vitro identification of a subset of patients with CU of possible autoimmune origin in which a third line therapy with immunomodulatory drugs could be contemplated (1, 2, 4). As regard, cyclosporine has been successfully employed in randomized controlled trials involving severe, unremitting AICU (25, 26) and its effectiveness may be attributed to the ability to inhibit basophil and mast cell degranulation as well as T-cell dependent antibody formation by B cells (25, 27–29). In our study we provide the first evidence that, in severe ASST+ CU patients who underwent short-term CyA treatment, serum-induced CD63 expression is significantly reduced during therapy, in accordance to clinical remission and ASST response, suggesting that the assay may also become a promising tool for monitoring the effectiveness of immunomodulatory drugs in AICU. Recently, the presence of a histamine-releasing factor specific for mast cells and not active on basophils, other than an anti-FcɛRI or anti-IgE autoantibody, has been documented by Sabroe et al. (6) in a small subset of CU patients with positive ASST (about 9%) and, in these cases, a negative serum-induced basophil activation test result should be expected. Furthermore, although many functional and structural similarities between basophils and mast cells exist, a FCM detection system using mast cell lines and/or chimeric cell lines expressing the human FcɛRI may be validated in the future for a larger screening of CU sera with both basophil and mast cells activating properties.