The Role Of Survivin In GM-CSF/G-CSF-induced Anti-apoptotic Effects On Terminally Differentiated Neutrophils

IntroductionNeutrophils are most abundant leukocytes in peripheral blood and immune system. They have a short lifespan and rapidly undergo spontaneous apoptosis to facilitate normal cell turnover and immune system homeostasis. Granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) are important pro-inflammatory cytokines in hematopoiesis. They can stimulate proliferation and differentiation of hematopoietic stem cells (HSC) and promote maturation of neutrophils. It is well known that these cytokines extend survival of neutrophils by delaying apoptosis. Although the anti-apoptitic effects of these cytokines have been shown to involve Bcl-2 family members including Mcl-1, Al and Bax, and the inhibitor of apoptosis protein (IAP) family members, such as cellular IAP-2, their anti-apoptotic mechanisms are not fully understood.Survivin is a novel member of the inhibitor of apoptosis protein family with a unique structure. Until now, five splice variants of survivin have been found in humans. It appears that different survivin isoforms have different subcellular localizations and different apoptosis regulatory functions as well. It was demonstrated in many investigations that survivin was highly expressed in most human tumor tissues and malignant cells. The higher expression of survivin was closely correlated with the poor prognosis of the patients, and the inhibition of survivin by using antisense oligonuleotides, small interfering RNAs and dominant negative mutants showed some anti-tumor effects. Recently, survivin was also found to be expressed in normal adult cells, suggesting its role in proliferation and survival of normal cells. Most recently, it was found that survivin was expressed in terminally differentiated neutrophils that were isolated from the patients with cystic fibrosis, a chronic inflammatory disease. The study also showed that pro-inflammatory cytokines, such as GM-CSF and G-CSF, induced survivin in normal neutrophils, and antisense-mediated inhibition of survivin expression prevented the anti-apoptotic effects of GM-CSF/G-CSF, suggesting that survivin might function on cytokine-induced anti-apoptosis with unknown mechanisms. In our experiment, we tried to investigate the role and mechanism of survivin in the cytokine-induced survival on terminally differentiated neutrophilis, which would contribute to the future research in the apoptitis regulation of neutrophils.MethodsIn this experiment, we used a human promyelocytic leukemia cell line, HL60, and freshly isolated normal peripheral blood neutrophils. To induce differentiation towards neutrophilic or monocytic granulocytes, HL60 cells were cultured with 1μmol/L of all-trans retinoic acid (ATRA) or 0.13μmol/L of phorbol 12-myristate 13-acetate (PMA) for 4 days. Differentiated HL60 and normal human neutrophils were stimulated with or without certain concentrations of GM-CSF/G-CSF for indicated periods. The survivin mRNA and protein expression were examined by real-time PCR SYBR Green method and Western Blotting, respectively. The neutrophis undergoing apoptosis were determined by annexin V-FITC/PI staining followed by flow cytometry analysis. And PCR products amplified from HL60 and neutrophil-derived mRNA were sequenced by the dideoxy chain-termination method.ResultsWith the differentiation of HL60 induced by ATRA and PMA, the survivin mRNA levels dramatically decreased and it became less than 1% of the initial level on day 4. Consistent with the change of survivin mRNA, the highly expressed survivin protein became undetectable on day 4. During the HL60 differentiation induced by ATRA, 10 ng/mL of GM-CSF significantly increased survivin expression, compared to those cells without GM-CSF treatment. In differentiated HL60, GM-CSF induced survivin expression time- and dose-dependently, and the maximum mRNA increase was obtained by 24-hour stimulation with 1 ng/mL of GM-CSF. In contrast, GM-CSF did not induce survivin expression in PMA-treated monocyte-like HL60 cells. However, normal neutrophils showed a 6-fold increase in survivin mRNA after a 24-hour culture in the absence of cytokines. The presence of 10 or 50 ng/mL of GM-CSF or 25 ng/mL of G-CSF induced no further elevation. There was no significant difference in survivin expression among groups treated with or without cytokines for 24 hours. In spite of the lack of survivin induction, GM-CSF and G-CSF exerted their anti-apoptotic effects on neutrophils, as demonstrated by the reduced apoptotic cell fractions from 5%～10% at 6 hour to 15%～20% at 24 hour. Further determination of survivin isoforms suggested that HL60 expressed wild-type survivin, while the isoform expressed in neutrophils was the pro-apoptotic survivin-2α.ConclusionIn our experiment, we found that survivin expression decreased with the differentiation of HL60. GM-CSF induced survivin in differentiated HL60 cultured with ATRA but not PMA. However, GM-CSF/G-CSF could not induce survivin in terminally differentiated normal neutrophils and the survivin isoforms are different between HL60 and neutrophils, which might explain their different responses to GM-CSF. Survivin is not involved in the GM-CSF/G-CSF-induced anti-apoptosis in neutrophils.