Astragalus polysaccharide promotes the release of mature granulocytes through the L-selectin signaling pathway
- 1.6k Downloads
This study aims to investigate the leukogenic effect of astragalus polysaccharide (APS), to compare its effect of increasing the numbers of mature granulocytes with that of granulocyte colony-stimulating factor (G-CSF), and to investigate the mechanism.
Rats were arbitrarily grouped into four groups (control, cyclophosphamide (CTX), CTX + APS, and CTX + G-CSF groups), and each group was then arbitrarily divided into five subgroups according to the time period since CTX infusion (0, 4, 7, 10, and 14 days). The expression of leukocyte selectin (L-selectin), its ligand, and shedding-related protease on granulocytes was analyzed. Leukocyte counts were obtained. Chemotactic capacity of polymorphonuclear leukocytes (PMNLs) was assessed.
Both APS and G-CSF restored the expression of L-selectin, P-selectin glycoprotein ligand-1 (PSGL-1), CD11b/CD18, and ADAM17 to normal levels (P > 0.05 vs. control group on each time point), with APS eliciting a greater effect than G-CSF (P = 0.005 on day 7, P < 0.001 on day 10 and 14 for L-selectin; P = 0.038 on day 7, P = 0.001 on day 10, P < 0.001 on day 14 for PSGL-1; P < 0.001 on day 7, 10 and 14 for ADAM17; P < 0.001 on day 7, 10, and 14 for CD11b/CD18). The percentages of the bands and segmented bone marrow (BM) cells in myeloid neutrophils were higher in the CTX + APS group than in the CTX group on day 7 (P = 0.030) and reached normal levels on day 10 (P = 0.547) and 14 (P = 0.431) vs. control group. The ability of APS to increase numbers of PMNLs in peripheral blood after chemotherapy was significantly superior to that of G-CSF 7 days after chemotherapy (P = 0.029 on day 10, P = 0.006 on day 14). Moreover, APS more significantly improved the chemotactic ability of PMNLs among mature BM granulocytes and peripheral blood neutrophils after chemotherapy than did G-CSF (P < 0.001 on day 7, P = 0.001 on day 10 and P = 0.005 on day 14).
APS promoted the differentiation and chemotactic ability of BM granulocytes via the L-selectin signaling pathway.
KeywordsBone Marrow Mean Fluorescence Intensity Total White Blood Cell Bone Marrow Smear Segmented Cell
Granulocyte colony-stimulating factor
Mean fluorescence intensity
P-selectin glycoprotein ligand-1
Zymosan-activated fetal bovine serum
Chemotherapy frequently causes neutropenia, which is a common and severe adverse effect that interrupts the treatment. Granulocyte colony-stimulating factor (G-CSF) is used to promote the proliferation of bone marrow (BM) hematopoietic cells as a treatment for neutropenia. However, apoptosis of hematopoietic progenitor cells and increased proliferation split times become evident after G-CSF stimulation . Moreover, BM does not have enough niches for the implantation of proliferated hematopoietic stem/progenitor cells; as a result, these cells may migrate directly to the peripheral blood and undergo apoptosis in the liver, spleen, and lung, causing a transient increase of leukocytes in blood . Furthermore, long-term and excessive administration of a stimulating factor can lead to increased expression of matrix metalloproteinase-9 (MMP-9) in CD34+ cells (naive hematopoietic cells), which dissolves the BM sinusoid matrix, thereby allowing proliferated hematopoietic stem/progenitor cells to move into the peripheral blood . The deformation, movement, and phagocytic abilities of CD34+ cells are weaker than those of mature cells, so the presence of these cells in peripheral blood results in a loss of anti-infection activities. Excessive release of naive cells can increase the incidence of leukemia . The long-term administration of stimulating factors may galvanize tumor cell invasion and angiogenesis .
In some young patients who undergo first-line chemotherapy, active myeloproliferation at 7 days after chemotherapy indicates that hematopoietic cells are not completely suppressed or that they recover (Additional file 1). Chemotherapy could hinder cell adhesion to the sinusoid epithelium, prevent release of BM polymorphonuclear leukocytes (PMNLs), and change the morphology and number of BM sinusoids . Therefore, failure of BM PMNLs release may also cause neutropenia.
We hypothesized that the interaction between leukocytes and vascular endothelial cells (ECs) when PMNLs were released from BM was similar to the interactions involved in the movement of blood PMNLs out of the capillaries. In such interactions between leukocytes and ECs, as selectin family member (i.e., leukocyte selectin; (L-selectin))  is involved in the initial attachment of circulating leukocytes to the endothelium. P-selectin glycoprotein ligand-1 (PSGL-1) on adherent neutrophils acts as a functional ligand for L-selectin . Afterwards, L-selectin is rapidly shed owing to the action of ADAM17, a proteolytic enzyme ; as a result, leukocytes roll on ECs. Neutrophils firmly adhere to ECs by subsequently upregulating CD11b/CD18  and move into sites of inflammation. L-selectin is then crosslinked with L-selectin on other leukocytes, resulting in increased levels of intracellular calcium, p38MAPK activation, and expression of mRNAs for TNF-α and IL-8 . These processes result in aggregation of PMNLs at sites of inflammation and degranulation in response to inflammation.
L-selectin may also be involved in the adhesion between granulocytes and sinusoid matrix ECs in the BM. A continuous increase in the expression of L-selectin and subsequent movement of mature cells from the BM stroma into the sinusoids, along with a gradual decrease in CD34 expression, were observed during the differentiation and maturation of BM blood cells . This process was also affected by similar ligands of adhesion molecules and endogenous proteolytic enzymes .
Astragalus polysaccharide (APS) has been shown to significantly increase the numbers of leukocytes, neutrophils, and platelets at 4, 7, 10, and 14 days after chemotherapy . The present study aims to investigate the leukogenic effect of APS, to compare its effect of increasing the numbers of mature granulocytes in the blood with that of G-CSF, and to investigate the underlying mechanism of action.
The experimental protocols were approved by the Animal Ethical and Welfare Committee of Tianjin Medical University Cancer Institute and Hospital (No. 058, date: 2013.1.7) (Additional file 2). The principles of laboratory animal care and the guidelines of the National Animal Welfare Law of China were followed when applicable.
Animal and pharmacological investigation on different drugs
Male Wistar rats (weighing 180–230 g) obtained from Charles River Laboratories (China; Certificate No: SCXK (jing) 2012–0001) were housed in cages (200 rats in 40 cages, five rats per cage) placed in temperature-controlled rooms. Rats received water and food ad libitum until they were needed for experimentation. The rats were arbitrarily grouped: control group, cyclophosphamide (CTX; Jiangsu Hengrui Medicine Co., Ltd., Jiangsu, China) group, CTX + APS (Tianjin Cinorch Pharmaceutical Co., Ltd., Tianjin, China) group, and CTX + G-CSF (Qilu Pharmaceutical Co., Ltd., Jinan, China) group. Fifty rats were included in each group. Each group was then arbitrarily divided into five subgroups (10 rats per subgroup) to provide samples on different numbers of days after CTX infusion (0, 4, 7, 10, and 14 d). Control animals received 0.4 mL of sterile phosphate-buffered saline (PBS, Gibco BRL Co.Ltd., Grand Island, NY, USA).
Drug doses and schedule of administration were chosen according to the results of previous studies: (1) intraperitoneal injection of 40 mg/kg CTX from day 0 to 1 ; (2) intravenous injection of 100 mg/kg APS from day −2 (2 days before CTX injection) to 11 ; and (3) subcutaneous injection of 10 μg/kg G-CSF from day 2 to 5 .
Blood and BM sampling
The rats were anesthetized by intraperitoneal injection of chloral hydrate at selected time intervals. Blood samples were drawn from the abdominal artery and placed in vacutainer tubes containing EDTA (BD Vacutainer Systems, Plymouth, UK). The rats were then sacrificed and their femurs were immediately removed. The epiphyses were cut transversely and the BM cells were flushed out with Iscove’s modified Dulbecco’s medium containing 584 mg/LL-glutamine (IMDM; HyClone; China) supplemented with 10 % heat-inactivated fetal calf serum (FCS; Yuanhengjinma Biotechnology Kaifa Co., China) and 100 U/mL lithium heparin. The sternum was removed and extruded for BM smears.
Fractionation of BM cells and isolation of blood PMNLs
Percoll media (GE Healthcare, Bio-Sciences AB) of different densities were prepared as previously described . A BM cell suspension or whole blood (3 mL) was gently layered on top of a two-layer discontinuous Percoll gradient: 1.093 and 1.077 g/mL (72 % and 60 % Percoll) densities for BM; 1.068 and 1.058 g/mL (51 % and 42 % Percoll) densities for blood. The tubes were then centrifuged at (1000 × g, 20 min, room temperature; Multifuge 1R, Thermo Fisher, USA) to separate the rat BM into three fractions. The middle-level cell fraction, which contained PMNLs, was washed twice in 10 mL of PBS (300 × g, 10 min, room temperature) to remove the remaining Percoll, and erythrocytes contaminating the pellet were removed using an Optilyse C lysis solution (Beckman Coulter, France). The PMNLs, separated by Percoll, were confirmed as BM neutrophil cell lines at various stages of differentiation by flow cytometry and microscopy using oil lens . We used only samples containing >90 % BM PMNLs (bands and segmented granulocytes) and circulating PMNLs with 95 % cell viability, as determined by trypan blue staining.
Expression of L-selectin, PSGL-1, CD11b/CD18, and ADAM-17 was assessed using a FACScan flow cytometer (Beckman Coulter EPICS-XI, USA). BM bands, segmented cells, and circulating PMNLs were gated in a forward scatter (cell size) vs. side scatter (granularity) dot plot. Mature BM granulocyte samples were pretreated with saturating concentrations of phycoerythrobilin (PE)-conjugated hamster anti-rat L-selectin monoclonal antibody (BD Pharmingen, Catalog No. 551398, USA) with mouse anti-rat PSGL-1 monoclonal antibody (KPL-1;Abcam, Catalog No. ab78188, UK), and PE-conjugated mouse anti-rat CD11b/CD18 monoclonal antibody (BD Pharmingen, Catalog No. 562105) with rabbit anti-rat ADAM17 polyclonal antibody (Abcam, Catalog No. ab2051). Fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit IgG and goat anti-mouse IgG (ZSGB-BIO, China) were used as secondary reagents. Negative controls were obtained by omitting monoclonal antibodies. Class-matched irrelevant PE-labeled hamster IgG2isotype control (BD Pharmingen, Catalog No. 553965) and FITC-labeled goat anti-mouse IgG were used to evaluate non-specific antibody binding. Data from 100,000 events per sample were acquired. The percentage of fluorescent cells (%F) was used to quantify L-selectin, PSGL-1, and ADAM17 expression. The mean fluorescence intensity (MFI) was used to quantify CD11b/CD18 expression after the neutrophils were gated according to their characteristic forward and side scatter properties.
Leukocyte countsand morphological analysis
The total leukocyte count in peripheral blood samples was obtained using an automated cell counter (MEK-6318 K, Dobio, Japan). Leukocyte differential counts were obtained for a minimum of 100 cells in blood and BM smears stained with Giemsa. Granulocytic cells were classified into immature granulocytes, mature granulocytes (bands and segmented), eosinophils, and mononuclear cells.
Preparation of zymosan-activated fetal bovine serum (ZAS)
ZAS was prepared as described previously . In brief, a bovine serum pool with 15 mg/mL of zymosan (Sigma-Aldrich, Switzerland, USA) was incubated at 37 °C for 60 min. The mixture was subsequently incubated at 56 °C for 30 min and cooled. Afterwards, the mixture was centrifuged at 1100 × g at 4 °C for 15 min. ZAS was diluted in a 1:5 Hanks balanced salt solution (without calcium and magnesium; Gibco BRL Co. Ltd., USA) and filtered through a 0.22-μm filter (Merck Millipore, Germany).
Measurement of chemotaxis
A micropore membrane was used to assess the chemotactic ability of mature BM granulocytes and blood PMNLs. Micropore inserts (diameter = 12 mm; pore size = 3 μm; Corning Incorporated, USA) were coated with collagen using a 0.1 % solution of calfskin collagen type I (Gibco, Invitrogen Corporation, Grand Island, USA). The cells were adjusted to 106/mL in PBS. For the treated group and the untreated control group, 200 μL of either PMNL suspension or mature BM granulocytes were added to the upper chambers. For the treated group, 400 μL of ZAS was placed in the lower chamber. For the control group, 400 μL of Hanks balanced salt solution was used. All group samples for the assays were incubated for 90 min at 37 °C in 95 % air and 5 % CO2. The migrated PMNLs and mature BM granulocytes were counted using a Burker chamber (Pyser-SGI, UK) at a magnification of 10 × 40. Results are expressed as the percentage of cells that migrated through the membrane. Each assay was performed in duplicate.
Data are expressed as means ± standard deviation (SD). All statistical analyses were performed in SPSS 17.0 (SPSS Inc., Chicago, IL, USA). Data were compared using the Kruskal-Wallis test. Dunn’s multiple contrast hypothesis test was used to compare each treatment group mean with each other group mean. The Mann–Whitney U test was used for unpaired observations. A P value less than 0.05 was considered significant. The effect of multiple comparisons was corrected for with the Bonferroni method.
HE staining of BM
Peripheral blood leukocyte and neutrophil count
Expression of L-selectin, PSGL-1, ADAM17, and CD11b/CD18
On day 4, the positive expression rate (F%) of L-selectin in mature BM granulocytes in the CTX group was 59.10 ± 8.45 %, which was higher than that in the control group (P < 0.001). F% decreased on days 7, 10, and 14. A significant difference was observed on days 7 and 14 (P < 0.001 on day 7 and P = 0.043 on day 14vs. control group), whereas F% in the APS group gradually increased from day 4 to 14 after chemotherapy and reached 49.53 ± 8.92 % on day 14. F% of L-selectin in the G-CSF group was the highest on day 4 (48.60 ± 3.08 %) and gradually decreased to 28.67 ± 9.10 % on day 14. In contrast, F% of L-selectin in the APS group was higher than that of the G-CSF group after 7 days (P = 0.005 on day 7, P < 0.001 on day 10, P < 0.001 on day 14). On day 4, F% of L-selectin in the two treatment groups was significantly lower than that in the CTX group (P < 0.001 vs. APS group, P = 0.001 vs. G-CSF group), but significantly higher than that in the CTX group after 7 days (P < 0.001 on day 7, P < 0.001 on day 10, P < 0.001 on day 14 vs. APS group; P < 0.001 on day 7, P = 0.001 on day 10, P = 0.003 on day 14 vs. G-CSF group).
F% of ADAM17 in mature BM granulocytes in the CTX group on days 4 to 14 was insignificantly lower than that in the control group (P = 0.176 on day 4, P = 0.204 on day 7, P = 0.101 on day 10, P = 0.368 on day 14). At each time point, the APS group showed higher F% than did the control, CTX, and G-CSF groups (P = 0.007 on day 4, P < 0.001 on day 7, P < 0.001 on day 10, P < 0.001 on day 14 vs. control group; P = 0.003 on day 4, P < 0.001 on day 7, P < 0.001 on day 10, P < 0.001 on day 14 vs. CTX group; P = 0.006 on day 4, P < 0.001 on day 7, P < 0.001 on day 10, P < 0.001 on day 14 vs. G-CSF group), with the maximum F% of 83.52 ± 12.36 % obtained 14 days after chemotherapy. F% of ADAM17 in the G-CSF group did not change significantly at each time point (P = 0.892 on day 4, P = 0.574 on day 7, P = 0.727 on day 10, P = 0.822 on day 14 vs. control group; P = 0.425 on day 4, P = 0.168 on day 7, P = 0.062 on day 10, P = 0.916 on day 14vs. CTX group; Fig. 3).
MFI of CD11b/CD18 on mature BM granulocytes significantly decreased after CTX treatment, and was further reduced by 50 % on days 10 and 14. The minimum MFI (15.98 ± 8.16) in the APS group was obtained on day 4; the MFI was increased significantly on days 7, 10, and 14 (P = 0.154 on day 7, P = 0.169 on day 10, P = 0.052 on day 14vs. control group, P = 0.028 on day 7, P < 0.001 on day 10, P < 0.001 on day 14vs. CTX group). The maximum MFI (29.56 ± 5.12) was obtained in the G-CSF group on day 4 (P = 0.136vs. control group). MFI in the G-CSF group was significantly decreased on days 7, 10, and 14 compared with that in the APS group (P < 0.001 on day 7, P < 0.001 on day 10, P < 0.001 on day 14). MFI in the G-CSF group was lower than that in the control group (P > 0.05) but insignificantly higher than that in the CTX group (P > 0.05) (Fig. 3).
Differentiation of BM granulocytes
Chemotactic ability of mature BM granulocytes and peripheral blood PMNLs
Design of treatment protocols
The administration of 10 μg/kg G-CSF from day 2 to day 5 after chemotherapy based on a previous report  and the results of our pilot experiment could enable the maximal stimulation of proliferation of neutrophils to be achieved, leading to severe leakage from vessels, edema, and even the death of several rats . APS was given in different ways in our pilot studies to obtain the maximal effect; ‘pre-chemotherapeutic administration’ and maintenance for 11 days after chemotherapy were determined to be the best protocols to increase neutrophils. The most suitable protocols for the two drugs were chosen to compare their maximal effects and to ensure safety in animals.
Effects on adhesion/release-related protein expression in mature BM granulocytes
Frenette and Weiss  confirmed that PMNLs expressing L-selectin can be preferentially released from the BM into the peripheral blood. ADAM17 is a major proteolytic enzyme that hydrolyzes the extracellular and juxtamembrane region of L-selectin . Our results showed that CTX could reduce expression of L-selectin, PSGL-1, CD11b/CD18, and ADAM17 from day 7–14 by interfering with initial binding, stable adhesion between granulocytes and sinusoidal endothelial cells, and smooth PMNL release. G-CSF could reduce this interference to a certain extent, but the effect was short lived; contrastingly, APS could significantly increase the expression levels of key proteins involved in granulocyte adhesion and release for a longer time (maximum of 7 days to 14 days). To our knowledge, our study is the first to elucidate that APS could relieve chemotherapy-induced impairment on key factors related to adhesion and release of PMNLs.
Effects on microvessel and sinusoid structures in BM
Chemotherapeutic agents could significantly reduce hematopoietic islets, resulting in severe suppression in bone marrow hematopoiesis . However, the reasons for chemotherapy-induced anemia should be more than just inhibition of proliferation of hematopoietic cells alone. Our findings revealed that CTX resulted in a fuzzy structure of microvessels and sinusoids in BM 4 days after chemotherapy. This damage in a hematopoietic microenvironment could impair the environment for proliferation and differentiation of hematopoietic cells, and also interfere with the adhesion and release of granulocytes. G-CSF could not restore the structure of microvessels and sinusoidal ECs. After APS treatment, the structures of BM microvessels and sinusoids became clear and organized, and adhesion and release of mature BM granulocytes were promoted. Given the enhancement of the immunocytotoxic function of APS against malignancies , we believe that its ‘protection’ of the bone marrow stroma could hardly protect tumors from chemotherapy.
Effects on the differentiation and release of granulocytes in BM
The BM section of the humerus showed that the BM cavity was filled with adipose cells 4 days after chemotherapy and erythroid and myeloid cell counts significantly decreased. Xue et al. [24, 25] found a severe defect in BM by loosing hematopoietic cells in VEGF tumor-bearing mice probably due to possibility for VEGF to mobilize BM hematopoietic stem cells to peripheral tissues and organs. After APS and G-CSF administration, the proliferation of myeloid cells was observed. In the G-CSF group, the ratio of mature BM granulocytes to the total neutrophils was lower than that of the CTX group, suggesting that G-CSF remarkably stimulated the proliferation of naive hematopoietic cells . Only 5 % of PMNLs were found 4 days after chemotherapy, when the highest levels of WBC were found in G-CSF group, suggesting that G-CSF prompts non-PMNLs releasing from bone marrow to peripheral blood. Janowska-Wieczorek et al.  demonstrated that G-CSF-induced the release of gelatinase B, which has a major function in the migration of BM early-stage myeloid cells into the blood. The total white blood cells reached normal level as induced by APS at day 14, while the proportion of mature BM granulocytes to the total neutrophils reached normal levels at day 10 and 14 in the APS group. Above results confirmed that APS promoted myeloid differentiation and maturation, and reduced mobilization of naïve BM hematopoietic cells to peripheral tissues, which yield a question that ought to be explored and might be the possibility for APS to restore BM hematopoiesis in patient with advanced cancer. APS augmented the expression of signalling molecules, releasing PMNLs after chemotherapy. This result is consistent with what is observed in leukemia cells with lower L-selectin expression, showing impaired transendothelial migration in vitro .
Effects on the chemotactic ability
To assess the chemotactic ability of PMNLs, we measured the ability of mature BM granulocytes and circulating PMNLs to pass through collagen-coated membranes to ZAS. The chemotactic ability of BM and PMNLs in the CTX group markedly decreased to 50 % of that in the control group at 14 d after chemotherapy. G-CSF could increase the chemotactic ability of PMNLs until day 7 after chemotherapy, but APS consistently increased this capability of PMNLs more effectively than G-CSF did. This result mightbe attributable to the upregulation of key proteins, such as TNF-α and IL-8, in the L-selectin signalling pathway in PNMLs by APS.
The activation of human granulocytes via cross-linking between L-selectin and its antibody can increase intracellular Ca2+ concentration , production and phosphorylation of superoxides , and activation of key signalling proteins . Cross-linking of L-selectin and its ligand as sulphated galactocerebroside increased the expression of TNF-α and IL-8 , causing granulocytes to penetrate the layer of human umbilical vein ECs. The inability of G-CSF to increase the chemotactic ability was probably due to its inability to restore the key proteins in the L-selectin signalling pathway. High expression of CD11b/CD18 was observed after the activation of granulocytes by cross-linking of L-selectin, which is closely related to the chemotactic ability of granulocytes in addition to mediating the stable adhesion of granulocytes .
APS promoted differentiation and chemotactic ability of BM granulocytes, involving the L-selectin signalling pathway.
The authors gratefully acknowledge Tianjin Cinorch Pharmaceutical Co., Ltd., for their utmost support in cordially providing the Astragalus polysaccharide. The authors would also like to thank the following individuals: Xi-ying Wei for his technical support in flow cytometry; Yong-ming Wang for his help on animal feed and technical assistance; and Hong-guang Ying and Ming-yun Zhao for their assistance on the morphological analysis of the bone marrow and peripheral blood granulocytes. This study was supported by grants from Tianjin Municipal Science and Technology Commission Application Research Project (No. 11JCYBJC11300 and No. 12ZCDSY15600 for Kai Li, No. 11JCZDJC18900 for Richeng Jiang), and the National Natural Science Foundation of China (No. 81372517 and No. 81000899 for Richeng Jiang).
- 10.Laudanna C, Constantin G, Baron P, Scarpini E, Scarlato G, Cabrini G, et al. Sulfatides trigger increase of cytosolic free calcium and enhanced expression of tumor necrosis factor-alpha and interleukin-8 mRNA in human neutrophils. Evidence for a role of L-selectin as a signaling molecule. J Biol Chem. 1994;269(6):4021–6.PubMedGoogle Scholar
- 13.Zhao MR, Zhou J. Effect of astragalus polysaccharide on suppression of bone marrow after chemotherapy in patients with malignant tumor [J]. Tianjin J Tradition Chinese Med. 2007;2:012.Google Scholar
- 15.Wang LC, Sun H, Li K. The effects of APS and G-CSF on bone marrow function after chemotherapy. Chin J Clin Oncol. 2008;35(23):1373–6.Google Scholar
- 30.Montecucco F, Steffens S, Burger F, Da Costa A, Bianchi G, Bertolotto M, et al. Tumor necrosis factor-alpha (TNF-α) induces integrin CD11b/CD18 (Mac-1) up-regulation and migration to the CC chemokine CCL3 (MIP-1α) on human neutrophils through defined signalling pathways. Cell Signal. 2008;20(3):557–68.PubMedCrossRefGoogle Scholar
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.