Morphological and Functional Characterization of CD34⁺cell-Derived Mast Cells from Sickle Cell Anemia Patients

Introduction: Mast cells (MC) activation has been described as a key factor on several disorders, but their role on sickle cell anemia (SCA) remains unclear. SCA is characterized by chronic inflammatory events orchestrated by activated leukocytes, which contribute to vaso-occlusive crisis. Physical interplay, release or production of mediators upon MC activation could affect the response of other cells. Stable MC and eosinophils (Eos) interplay and bidirectional modulation has been described on allergic and inflammatory diseases. We aimed to determine whether MC obtained from CD34⁺ stem cells of SCA patients, compared to healthy volunteers (HV), show differences in phenotype, function, or ability to interact with Eos.

Methods: CD34⁺cellswere isolated from peripheral blood (HV: 60 ml; SCA: 30 ml) and cultured in StemPro34® medium with glutamine (2mM), LDL (10 mg/ml), SCF, IL-6 and IL-3 (100 ng/ml each). Culture was weekly expanded (medium without IL-3); differentiation was confirmed by toluidine blue staining and FACS (CD117⁺, FceRI⁺). After 4 weeks, surface marker expression of MC differentiation/activation, adhesion and mast cell/eosinophil related of HV and SCA was compared by FACS. Imaging flow cytometry (IFC) was used to define nuclear morphology, to identify differences in other marker expression and to characterize in vitro physical contact between MC and Eos isolated from peripheral blood from HV and SCA individuals. Cells were separately marked with fluorescent, lipophilic stains, and Feature Finder tool was used to pinpoint frequency and remarkable features for heterotypic conjugates formation. Standardized conditions were a 1:3 MC-Eos ratio, incubation for 5 minutes at 37°C and plotting Shape Ratio and Circularity features to quantify the occurrence of MC-Eos conjugates. In addition, cells were stained with fluorescent antibodies after 2 hours of interaction to evaluate the changes in marker expression during this process.

Results: There was not difference in CD34⁺ cell count between HV and SCA individuals (p=0.848); however, a higher number of MC was obtained from SCA samples (4^th week) (HV: 1.67x10⁵±0.40; SCA: 4.38x10⁵±1.22; p=0.027). Mature SCA from HV (n=10) and SCA (n=10) were obtained after 5 weeks (CD117⁺, FceRI⁺>50%). FACS showed higher percentage of CD117⁺ cells in SCA cultures (HV 18.35±5.83%; SCA 65.25±12.28%; p<0.01). Regarding CD244 and Siglec-8, SCA samples showed higher positivity, although not statistically significant because of marked variation among individuals (CD244: HV 3.32±0.84%, SCA 16.30±10.89%; Siglec-8: HV 5.94±2.48%, SCA 8.8±5.32%, p>0.05). No difference in adhesion molecules expression was detected (data not shown). IFC analysis revealed no difference in nuclear morphology, p=0.352). Differences in CD117 and Siglec-8 expression allowed a clear separation of HV- and SCD-MC (Bkgd Mean CD117: RD 2.78; Bkgd Mean Siglec8 RD: 1.84). Heterotypical (MC-Eos) conjugates formation was a rare event (less than 5% of images); there was no difference in the percentage of conjugates (HV-MC/HV-Eos: 1.61±0.37%; HV-MC/SCA-Eos: 1.92±0.37%; SCA-MC/HVEos: 1.12±0.31%; SCA-MC/SCA-Eos: 1.26±0.27%; p>0.05). Analysis of marker expression after interaction showed that HV-MC changed CD48, CD63 and CD117 expression patterns in response to SCA-Eos challenge (Bkgd Mean RD: CD48 1.09; CD63 1.16; CD117, 1.31); similarly, SCA-MC co-cultures with SCA-Eos not only modified CD48 and CD63 (Bkgd Mean RD values: BV421-CD48 2.32; PB-CD63 2.27), but also altered CD193, in a lesser extent (Bkgd Mean CD193: RD 0.88).

Conclusion: We report for the first time the morphological and functional characteristics of CD34⁺-derived MC obtained from samples of SCA in comparison with HV. Our data suggest that MC from SCA patients may have a different expression profile of markers related to activation even after being withdrawn from exposure to chronic inflammation and vaso-occlusion in vivo . Surface markers corresponding to CD117, CD48, CD63 and CD193 in SCA animal models could be helpful to assess the activation state of MC and their response to experimental treatments targeting these cells.