Protective effects of cytokine combinations against the apoptotic activity of glucocorticoids on CD34+ hematopoietic stem/progenitor cells

Abstract

Haematopoietic stem cells can self-renew and produce progenitor cells, which have a high proliferation capacity. Chemotherapeutic drugs are toxic to normal cells as well as cancer cells, and glucocorticoids (GCs), which are essential drugs for many chemotherapeutic protocols, efficiently induce apoptosis not only in malignant cells but also in normal haematopoietic cells. Studies have shown that haematopoietic cytokines can prevent the apoptosis induced by chemotherapy and decrease the toxic effects of these drugs. However, the apoptosis induction mechanism of GCs in CD34⁺ haematopoietic cells and the anti-apoptotic effects of cytokines have not been well elucidated. In this study, we investigated the apoptotic effects of GCs on CD34⁺, a haematopoietic stem/progenitor cell (HSPC) population, and demonstrated the protective effects of haematopoietic cytokines. We used a cytokine cocktail containing early-acting cytokines, namely, interleukin-3 (IL-3), thrombopoietin, stem cell factor and flt3/flk2 ligand, and dexamethasone and prednisolone were used as GCs. Apoptotic mechanisms were assessed by immunohistochemical staining and quantified using H-scoring. Dexamethasone and prednisolone induced apoptosis in CD34⁺ HSPCs. GC treatment caused a significant increase in apoptotic Fas, caspase-3, cytochrome c and Bax, but a significant decrease in anti-apoptotic Bcl-2. Furthermore, as expected, cytokines caused a significant decrease in all apoptotic markers and a significant increase in Bcl-2. Thus, our findings suggest that CD34⁺ HSPCs are an extremely sensitive target for GCs and that cytokines protect these cells from GC-induced apoptosis.

Keywords: Apoptosis; CD34+ haematopoietic stem/progenitor cells; Cytokines; Glucocorticoids.

Fig. 1

The purity of enriched cells were determined by flow cytometry, after isolation cells were incubated with fluorescein isothiocyanate (FITC) conjugated mouse antihuman CD34 monoclonal antibody. The purity of the CD34⁺ cells was between 81 and 93.4% as seen in sample above. (Control refers to the group incubated only with serum free medium (SFC)). (UL: Upper Left; UR: Upper Right; LL: Lower Left and LR: Lower Right)

Fig. 2

Determining degree of peroxidase reaction in CD34⁺ hematopoietic cells. (+++) cells stained at intensity 3, (++) cells stained at intensity 2, (+) cells stained at intensity 1, (−) no reaction

Fig. 3

Peroxidase reaction levels calculated using H-score between serum free medium control and cytokine control that including IL3, SCF, Tpo and FL. The results represent the means ±SD of five individual experiments performed with cells from different donors. *p < 0.05

Fig. 4

Peroxidase reaction levels calculated using H-score in cells stained with apoptotic markers. a Reaction levels for Fas. b Reaction levels for Caspase 3. c Reaction levels for cytochrome c. d Reaction levels for Bax show expression levels of apoptotic proteins. (SCF) Serum free control, (CC) Cytokine control, (CD) Cytokine-dexamethasone, (D) Dexamethasone, (CP) Cytokine-prednisolone, (P) Prednisolone represent experiment and control groups. The results represent the means ± SD of five individual experiments performed with cells from different donors. *, **, ***, ****p < 0.05. More asterisk were used for comparison between different groups, but statistically significant was same value

Fig. 5

Peroxidase reaction levels calculated using H-score for antiapoptotic Bcl-2. (SCF) Serum free control, (CC) Cytokine control, (CD) Cytokine-dexamethasone, (D) Dexamethasone, (CP) Cytokine-prednisolone, (P) Prednisolone. The results represent the means ± SD of five individual experiments. *, **, ***, ****p < 0.05. More asterisk were used for comparison different groups, but statistically significant was same value