G-CSF Promotes Splenic Erythropoiesis Through A Renal Erythropoietin-dependent Mechanism In Mice

IntroductionGranulocyte colony stimulating factor (G-CSF) is an important cytokine in the body; G-CSF can promote the formation of granulocyte and enhance the activity of granulocyte. G-CSF is widely used for the treatment of various causes of neutropenia; G-CSF also has been routinely used to therapeutically mobilize hematopoietic stem and progenitor cells (HSPCs) to the bloodstream for transplantation. Recent reports suggest that G-CSF can promote tumor metastasis and worsen anemia in early breast cancer patients treated with chemotherapy agents. G-CSF administration leads to the healthy donor hematopoietic stem cell gene abnormal expression in a long time. Long term use of G-CSF in the treatment of congenital neutropenia may increase the risk of leukemia. Previously, we found that the secretion of G-CSF in breast cancer cells can induce erythropoiesis disorders. The medullar erythropoiesis is impaired, and the splenic erythropoiesis is enhanced in 4T1 tumor-bearing mice. Erythropoietin is the key cytokine in the production of red blood cells. The secretion of EPO is mainly depends on renal tubular interstitial cells. The functions of EPO and kindney in G-CSF-induced splenic erythropoiesis remain to be elucidated. To get a better insight into the link between G-CSF, erythropoiesis, anemia and cancer, we examined the roles of EPO and kidney in G-CSF-induced splenic erythropoiesis. Our study will make us to understand the pharmacological activities and adverse reactions of G-CSF better, help us to clarify the relationship between G-CSF and the occurrence and development of cancer, provide an important reference for clinical rational drug use and tumor therapy. At the same time, we can expand our understanding of the function of the spleen, which is conducive to understand the regulation mechanism of the body’s erythropoietic system.ObjectiveInvestigate the regulatory effects of G-CSF on medullar and splenic erythropoiesi and evaluate the functions of EPO and kindney in G-CSF-induced splenic erythropoiesis.Methods1. Mice were injected twice daily subcutaneously with recombinant human G-CSF or saline for 9 consecutive days. Bone marrow cells and splenocytes were harvested; the cells were incubated with FITC Rat Anti-Mouse Ter119(or APC Rat Anti-Mouse Ter119) and PE Rat Anti-Mouse CD71 antibodies. After staining, flow cytometry was performed for the quantification of CD71+Ter119+ cells.2. Bone marrow cells and splenocytes were isolated from control mice and mice treated with G-CSF; the cells were incutated with APC-conjugated anti-Mouse Ter119 antibody and thiazole orange. The reticulocytes (Ter119+Thiazole orange+) and erythroblasts (Ter119+Thiazole orange++) were analyzed by flow cytometry.3. Mice were treated with G-CSF for 9 consecutive days. Bone marrow and peripheral blood cells were made into cell smears. After the Wright-Giemsa staining, granulocyte and erythrocyte cell smears were observed under microscope.4. Splenocytes were isolated from control and G-CSF-treated mice. Then, nucleated splenocytes were plated in methylcellulose media according to the instructions of manufacturer. CFU-E colonies were counted under a microscope on days 7 of culture.5. Control and G-CSF-treated mice were anesthetized with sodium pentobarbital. Blood was collected by cardiac puncture and allowed to clot at room temperature for 1.5 h. Determination of serum concentrations of EPO was carried out using commercially available enzyme-linked immunosorbent assay kits, according to the manufacture’s instructions.6. EPO mRNA was detected in kidney by quantitative reverse-transcribed polymerase chain reaction (RT-PCR).7. Erythropoietin was administered to mice by daily subcutaneous injection for 4 consecutive days. The splenocytes were isolated and incubated with FITC Rat Anti-Mouse Ter119 (or APC Rat Anti-Mouse Ter119) and PE Rat Anti-Mouse CD71 antibodies. After staining, flow cytometry was performed for the quantification of CD71+Ter119+cells.8. The splenocytes were isolated from control and EPO-treated mice. The cells were incutated with APC-conjugated anti-Mouse Ter119 antibody and thiazole orange. The reticulocytes (Ter119+Thiazole orange+) and erythroblasts (Ter119+Thiazole orange++) were analyzed by flow cytometry.9. After G-CSF injection, recipient mice were transfused via tail vein injection with 150 μl (equivalent of 150 μl blood) of RBCs on days 2,4,6 and 8. The splenocytes were isolated and incubated with APC Rat Anti-Mouse Ter119 and PE Rat Anti-Mouse CD71 antibodies. After staining, flow cytometry was performed for the quantification of CD71+Ter119+ cells. The cells were incutated with APC-conjugated anti-Mouse Terl 19 antibody and thiazole orange; the reticulocytes (Ter119+Thiazole orange+) and erythroblasts (Ter119+Thiazole orange++) were analyzed by flow cytometry.10. Mice were treated with G-CSF for 9 consecutive days. Anti-EPO antibody was administered daily via intraperitoneal injection from day 4. The splenocytes were isolated and incubated with FITC Rat Anti-Mouse Ter119 (or APC Rat Anti-Mouse Ter119) and PE Rat Anti-Mouse CD71 antibodies. After staining, flow cytometry was performed for the quantification of CD71+Ter119+ cells. The cells were incutated with APC-conjugated anti-Mouse Ter119 antibody and thiazole orange; the reticulocytes (Ter119+Thiazole orange+) and erythroblasts (Ter119+Thiazole orange++) were analyzed by flow cytometry.11. Adenine-fed mice were treated with G-CSF for 9 consecutive days. The splenocytes were isolated and incubated with APC Rat Anti-Mouse Terl 19 and PE Rat Anti-Mouse CD71 antibodies. After staining, flow cytometry was performed for the quantification of CD71+Ter119+ cells. The cells were incutated with APC-conjugated anti-Mouse Ter119 antibody and thiazole orange; the reticulocytes (Ter119+Thiazole orange+) and erythroblasts (Ter119+Thiazole orange++) were analyzed by flow cytometry.12. After G-CSF injection, bilateral nephrectomy was performed on day 7. Splenocytes were harvested on day 9; the cells were incubated with APC Rat Anti-Mouse Ter119 and PE Rat Anti-Mouse CD71 antibodies. After staining, flow cytometry was performed for the quantification of CD71+Ter119+ cells. The cells were incutated with APC-conjugated anti-Mouse Ter119 antibody and thiazole orange; the reticulocytes (Ter119+Thiazole orange+) and erythroblasts (Ter119+Thiazole orange++) were analyzed by flow cytometry.13. HIF-la and HIF-2a protein levels of kidney were detected by western blot.Results1. G-CSF treatment impairs medullar erythropoiesis and promotes splenic erythropoiesis in mice.The long bones of the G-CSF-treated mice were paler than those of control mice, which were associated with a reduction in the total number of cells in one femur. The proportion of CD71+Ter119+ cells in G-CSF-treated mice was significantly lower than in control mice. G-CSF decreases reticulocytes and erythroblasts of bone marrow in mice. The results indicate G-CSF impaired the erythroblast terminal differentiation in bone marrow. G-CSF administration led to the increase the granulocytes of bone marrow and peripheral blood. G-CSF administration induced splenomegaly and increased the total cells in spleen. G-CSF treatment increased the numbers of CFU-E derived colonies from the spleen. G-CSF administration led to the increase of CD71+Ter119+ cell population of spleen in mice. G-CSF increased reticulocytes and erythroblasts of spleen in mice.2. G-CSF treatment induces high EPO secretion in kidney.G-CSF administration resulted in elevation of serum EPO concentrations and upregulation of EPO mRNA in kidney. G-CSF treatment led to high Hypoxia inducible factor-1,2α(HIF-1,2α) levels in kidney. EPO injections enhanced the development of erythroid cells in the spleen, and increased proportions of reticulocytes and erythroblasts in the spleens of EPO treated mice. Red blood cells transfusion decreased serum EPO concentrations, and normalized the development of erythroid cells, proportions of reticulocytes and erythroblasts in the spleen from G-CSF treated mice. EPO antibody inhibits G-CSF-induced splenic erythropoiesis.3. Renal dysfunction disrupts G-CSF-induced splenic erythropoiesisAdenine feeding induced renal dysfunction and decreased serum EPO levels in G-CSF-treated mice. Adenine feeding inhibited the splenomegaly induced by G-CSF. Adenine-exposed mice display repression of the development of erythroid cells in the spleen. Adenine feeding decreased the proportions of reticulocytes and erythroblasts in the spleen from G-CSF-treated mice. Bilateral nephrectomy decreased serum EPO levels in G-CSF-treated mice and prevented the splenomegaly induced by G-CSF. Bilateral nephrectomy repressed of the development of erythroid cells induced by G-CSF in the spleen. Bilateral nephrectomy decreased the proportions of reticulocytes and erythroblasts in the spleen from G-CSF-treated mice.ConclusionG-CSF promotes splenic erythropoiesis of mice by increasing the erythropoietin secretion in kidney.