CD25 as a unique marker on human basophils in stable-mildly symptomatic allergic asthma

Abstract

Background: Basophils in acute asthma exacerbation are activated as evidenced by their increased expression levels of activation markers such as CD203c and CD63. However, whether basophils of allergic asthmatics who are in stable phase and have no asthma exacerbations display a specific and distinctive phenotype from those of healthy individuals has yet to be well characterized.

Objective: We aimed to identify the phenotype of basophils from allergic asthmatics in the stable phase and investigate whether such a phenotype is affected by ex vivo allergen stimulation.

Methods: We determined by flow cytometry, the expression of surface proteins such as CD25, CD32, CD63, CD69, CD203c, and CD300a and intracellular anti-apoptotic proteins BCL-2, BCL-xL, and MCL-1. We investigated these markers in blood basophils obtained from well-characterized patients with stable-mildly symptomatic form of allergic asthma with no asthma exacerbation and from healthy individuals. Moreover, we determined ex vivo CD63, CD69, and CD25 on blood basophils from stable-mildly symptomatic allergic asthmatics upon allergen stimulation.

Results: In contrast to all tested markers, CD25 was significantly increased on circulating basophils in the patient cohort with stable-mildly symptomatic allergic asthma than in healthy controls. The expression levels of CD25 on blood basophils showed a tendency to positively correlate with FeNO levels. Notably, CD25 expression was not affected by ex vivo allergen stimulation of blood basophils from stable-mildly symptomatic allergic asthma patients.

Conclusion: Our data identifies CD25 as a unique marker on blood basophils of the stable phase of allergic asthma but not of asthma exacerbation as mimicked by ex vivo allergen stimulation.

Keywords: CD25; basophils; ex vivo stimulation; immunophenotype; stable-mildly symptomatic allergic asthma.

Figure 1

Relationship between expression levels of CD25 on basophils and FeNO levels in stable-mildly symptomatic allergic asthma patients. (A) Representative histogram showing CD25 expression level (MFI) on blood basophils in stable-mildly symptomatic allergic asthma patient (gray area) compared to healthy control (solid line) and FMO control (dotted line). (B, D-H) The expression levels of surface markers on blood basophils of stable-mildly symptomatic allergic asthma patients for the first (n=18) and second visit (n ≤ 11) compared to healthy controls (n≥9) measured by flow cytometry. Data are represented as mean ± SEM and reported as ΔMFI (MFI of test-MFI of FMO control) for CD25, CD63, CD69 and as MFI for CD32, CD203c and CD300a. Results were analyzed by two-tailed unpaired Mann-Whitney test.**P=0.0065, *P=0.0159, ns=non-significant (C) The relative percentage of blood basophils (dual positive for CD123 and CCR3) from stable-mildly symptomatic allergic asthma patients during their first (n=18) and second (n=11) visits compared to healthy subjects (n=10) (I) Correlation between CD25 expression levels (ΔMFI) and FeNO levels (ppb) in stable-mildly symptomatic allergic asthma patients of the first (left, n=18) and second visit (right, n=11). Spearman`s rank test was used for correlation analysis. Spearman coefficient (r) and level of significance (P) are indicated within the graph. (J) Surface expression levels of CD25 (ΔMFI) on blood basophils between <25 ppb (n=20) and ≥25 ppb FeNO level (n= 9) groups within stable-mildly symptomatic allergic asthma patients. Data are represented as mean ± SEM and analyzed using two-tailed unpaired Mann-Whitney test. *P<0.05. (B-J) Every patient is represented with specific symbol, while healthy controls are shown in closed black symbols. Patients who had both 1^st and 2^nd visits are marked in purple and those who had only 1^st visit are marked in green color. MFI, median fluorescence intensity; FMO, fluorescence minus one; Healthy, healthy control subjects.

Figure 2

Effects of ex vivo allergen challenge on CD63, CD69, and CD25 expression in activated and non-activated basophils. Whole blood samples from stable-mildly symptomatic allergic asthma patients (n=3) were stimulated with their respective allergen (20ng/ml or 100 ng/ml) or anti-FcεRI cross-linking antibody 29C6 (100 ng/ml). Unstimulated controls were exposed to stimulation buffer only. (A) Percentage of CD63^pos basophils in unstimulated controls, 20 ng/ml, and 100 ng/ml allergen and anti-FcεRI cross-linking antibody stimulated samples. (B, C) MFI of CD69 (B) and ΔMFI (MFI of test-MFI of FMO control) of CD25 (C) are shown on CD63^neg (open symbols) and CD63^pos basophils (closed symbols). (A-C) Patients 1, 2, and 3 are represented as circle, square, and triangle symbols, respectively. Data are shown as mean ± SEM and analyzed using one-way ordinary ANOVA tests, with Dunnett`s (A) or Bonferroni`s (B, C) multiple group comparison test. **P<0.01 and *P<0.05. ns, not significant.