Autoimmune chronic urticaria: new insights in diagnostic methods and pathogenetic mechanisms

Chronic urticaria (CU) is a common cutaneous inflammatory syndrome with a complex physiopathology. Although not life threatening, the disease has a deep impact on the patient’s quality of life. A particular subset of patients has autoimmune chronic urticaria (AICU), characterized by the presence of circulating anti-FcRI autoantibodies and by a more severe clinical behavior, often requiring aggressive therapies. The autologous serum skin test (ASST) is commonly used in clinical practice to identify the AICU patients, but its sensitivity and specificity are not fully satisfying. Many methods exist to detect in vitro anti-FcRI antibodies, but unfortunately they suffer of many limitations and not routinely available everywhere. For this reason, in recent years a basophil activation test (BAT) on flow-cytometry has been developed and is raising particular interest for its sensitivity and specificity, reproducibility and potential wide diffusion. Therefore we intended to set this technique, evaluating the surface expression of the activation markers CD63 and CD203c after stimulation of pools of basophils derived from atopic donors with sera of 20 CU patients and positive (fMLP and anti IgE) and negative controls (resting and 8 healthy patients sera). Moreover we explored changes in CD11b surface expression and influence of plasma stimulation on all the markers’ expression. More in detail, BAT cellular substrate derived from buffy coats pooled from K2-EDTA anti-coagulated peripheral blood of 4 atopic donors. Human albumin was added to the cellular pool to obtain a final albumin dilution of 0.2%. Thereafter, washing buffer alone, fMLP (as IgE-independent agonist) and anti-human monoclonal IgE were added in different tubes to the cellular suspension, in order to determine the basal level and the positive controls for the surface expression of the activation markers under evaluation; moreover 100l of sera and plasma were added in different tubes to the cellular suspension, in order to analyze their influence on markers expression. Three consecutive tests were performed on donors’ basophils with each stimulating agent. For better definition, undiluted sera and plasma were tested both after heat inactivation (left at room temperature and then heated to 56°C for 30 minutes to denaturate IgE and inactivate complement) and without heat inactivation. All samples were incubated for 20 minutes to 37°C. Basophils were evaluated on six colour flow cytometry, after monoclonal antibody staining and erythrocyte lysis. Basophils were gated as low SSC, HLA-DRnot-expressing and CD123bright cells; basophil surface markers CD63, CD203c and CD11b were evaluated by determining the levels of mean fluorescence intensity (MFI) for the three of them and the percentage of cells shifting to high expression only for CD63 (CD63%). All data were analyzed with adequate statistical tests. All CU patients were thoroughly examined at baseline, by evaluation of multiple clinical and laboratory parameters, and screened with ASST and autologous plasma skin test (APST), in order to identify AICU subjects. In particular, disease severity and impact on quality of life were measured by specific validated tools (US and CU2QoL, respectively). Our CU study population resulted overall consistent with the previously published data as regards female prevalence, mean age, disease duration, low basophil count, inflammation markers values and auto-antibody positivity. Moreover ASST gave more frequently positive result in female patients with higher disease severity, lower basophil count and IgE values, higher auto-antibody positivity (anti-nuclear and anti-thyroid), but failed to show correlation with longer disease duration, higher impact on quality of life, or higher inflammation markers values. Finally, ASST positivity resulted lower in our population as compared with literature data, but it is consistent with our clinical experience. APST positivity was higher than ASST positivity as expected, with good concordance between these two in vivo tests both for outcome and wheal reaction diameters. As regards flow cytometry analysis, assay reproducibility wasn’t negatively affected by the use of different atopic-donors pools as basophil source. The experimental conditions proved to be good, because we obtained lower unspecific activation levels than those reported in literature, whereas the activation marker expression levels after activating stimuli were as high as in previously published studies. Heat-inactivated sera determined higher values of markers expression as compared with matched not heat-inactivated sera, thus determining higher diagnostic efficiency. We were able to confirm a positive correlation between ASST positive result and higher values of CD63%, CD63MFI and CD203cMFI expression. Overall, our flow cytometry assay results indicate that CD203c has higher sensitivity, CD63 has higher specificity, and diagnostic efficiency can be improved when CD63% and CD203cMFI results are combined. Moreover flow cytometry BAT gave more frequently positive result in patients with higher disease severity, lower basophil count and IgE values, higher auto-antibody positivity (anti-nuclear and anti-thyroid), although without statistically significant differences in many cases. In our assay, CD11b expression showed no modifications on atopic basophils after stimulation with tested agents, neither IgE-dependent nor IgE-independent; therefore it seemed to be not useful in BAT applied to the study of CU under these particular conditions. Reasons for this unexpected result are not clear, but reliability of our results is supported by the demonstration of a significant increase in CD11b expression on neutrophils after stimulation with fMLP and with sera from controls and ASST positive subjects under the same experimental conditions. Finally we provided new evidences that plasma does not exert a stimulatory effect on basophils in vitro. These results were unexpected because many observations suggested a theoretical higher stimulatory effect for plasma as compared with serum, although other authors recently showed that serum and plasma were comparable for skin testing but serum was better than plasma for in vitro assays; indeed, both specimens induced histamine release from donor basophils, but serum gave more frequently positive results. The reason for this difference is not clear, although the presence of citrate in plasma may increase the threshold necessary for histamine release from donor basophils. In particular, in our assay basophils exposure to plasma determined a decrease in CD63 expression and a slight increase in CD203c expression; both these changes were more pronounced for plasma from ASST+ CU patients. Thus, these results further underline the differences between CD63 and CD203c as activation markers and recall the possible involvement of plasma coagulation factors in urticaria pathogenesis.