The Eotaxin/CCR3 Axis And The Migration And Differentiation Of Bone Marrow CD34～+ Progenitors In Allergic Mice

IntroductionAsthma is a complex disorder characterized by reversible airway obstruction, bronchial hyperresponsiveness and airway inflammation. Asthma and mouse models of allergic respiratory inflammation are invariably associated with a pulmonary eosinophilia. It has been shown that eosinophils arise from CD34⁺ pluripotent stem cells in bone marrow, then they are released into the circulation. To support the recruitment of eosinophils into the tissues, there is an early release of eosinophils into the circulation, and subsequently an enhanced production of these cells within the bone marrow. Chemokines play an important role in accumulation of eosinophils at allergic inflammatory sites. Eotaxin is a chemokine implicated in eosinophil trafficking. The specific activity of eotaxin is mediated by the selective expression of the eotaxin receptor, CC chemokine receptor 3 (CCR3), which is the principal chemokine recepter on the surface of eosinophils. Furthermore, there is increasing evidence that in atopic subjects after exposure to allergen, distinct phenotypic changes occur within the BM progenitor cell population that, in addition to triggering the activation of eosinopoiesis, may also induce the migration of progenitor cells from theBM to the site of allergic inflammation. These progenitor cells may undergo in situ differentiation in the presence of locally elaborated cytokines and provide an ongoing source of inflammatory effector cells. It was reported, CCR3 is also expressed on human CD34⁺ progenitors , and its expression on CD34⁺ progenitors significantly increases in asthmatics. However, the effect of eotaxin/CCR3 in the process of progenitor differentiation and multiply is still not very clear. We hypothesized that eotaxin/CCR3 is involved not only in the migration of eosinophil but also in the migration and differentiation of CD34⁺ progenitor. So we observed the changes of eosinophils, CD34⁺ progenitors and CD34⁺/CCR3⁺ progenitors in a murine model of asthma, in order to investigate the effect of eotaxin/CCR3 on the migration of eosinophils and CD34⁺ progenitors. We administrated asthmatic mice with CCR3 mAb in vivo and in vitro chemotactic assays and colony-forming assay were performed to assess the effect of eotaxin/CCR3 on the migration and differentiation of CD34⁺ progenitors.Inhaled Glucocorticoids (GCCs) are the preferred anti-inflammatory therapy for the treatment of persistent asthma as recommended by international guidelines. GCCs have powerful anti-inflammatory effects by virtue of inhibiting pro-inflammatory cell recruitment and down-regulating the production of pro-inflammatory cytokines. The inhibitory effects of GCCs on the production of eosinophil-active Chemokines, such as eotaxin, and the effects of GCCs on expression of chemokine receptors in eosinophils per se, have been well established. In recent years, studies have shown that the migration of progenitor cells from the BM to the site of allergic inflammation and the differentiation in situ were involved in pulmonary eosinophilia in asthma. However, few data existed regarding the effects of GCCs on expression of chemokine receptors in the bone marrow progenitor cells and the effects on the migration of progenitor cells from the bone marrow to the site of allergic inflammation.We hypothesized that GCCs inhibit the expression of CCR3 in the bone marrow progenitors and also inhibit the chemotaxis of eotaxin on the bone marrow progenitors. In an attempt to address this hypothesis, we used a well-established mouse model of allergen-induced airway inflammation. For the preventionexperiment, mice were treated with an intraperitoneal injection of dexamethasone 1 hr before each pulmonary challenge.Purification and enrichment of CD34⁺ cells from asthmatic mouse bone marrow is required for this study. At present, there are different purification systems have been developed to isolate CD34⁺ cells, including CELLector flasks , immunoaffinity columns (Ceprate LC), immunomagnetic beads (Dynabeads, Baxter Isolex 50) , magnetic activated cell sorting (MACS) and Fluorescence Activated Cell Sorting (FACS). Previous studies indicated that the most reliable methods of purification for all human samples were fluorescence activated cell sorting (FACS) and magnetic activated cell sorting (MACS) which consistently yielded high purities (> 70%) and enrichment of colony forming cells (CFC). However, whether these methods are also feasible for separation of CD34⁺ cells from mouse bone marrow is not very clear up to now. In this context, we assessed the efficiency of magnetic activated cell sorting (MACS) separation in purification and enrichment of CD34⁺ cells from mouse bone marrow as the first step of this study.Part 1 Purification and enrichment of CD34⁺ cells from mouse bonemarrow using magnetic activated cell sorting (MACS)Objective: To assess the efficiency of magnetic activated cell sorting (MACS) separation in purification and enrichment of CD34⁺ cells from asthmatic mouse bone marrow.Methods: Male C57BL/6 mice, 6-8 week old, were sensitized two times and challenged three times by ovalbumin (OVA) as OVA/OVA group, and mice were treated by normal saline as control group (NS/NS group). Then mice bone marrow cells were harvested from the femurs and then separated by Percoll density gradient centrifugation to harvest Low-density mononuclear cells. Monocytes were removed by incubation in plastic flasks for 2 hr at 37℃ in an atmosphere containing 5% CO₂. Non-adherent mononuclear cells were harvest. CD34⁺ progenitor cells were purifiedsequentially from non-adherent mononuclear cell using a magnetic cell-sorting system. Three strategies were used, positive selection, negative selection and negative selection combined with positive selection. Flow cytometry was employed to determine the percentages of CD34⁺ cells before and after the purification using MACS with three strategies. The cell viability was assessed by means of trypan blue straining.Results: Slight higher percentages of CD34⁺ cells in bone marrow non-adherent mononuclear cell was found in asthma group mice than those in control group mice before purification [(2.98±0.62)% vs (1.79±0.75)%, respectively. P>0.05]. After positive selection, the purity of CD34⁺ cells harvest from asthma group mice is similar to those from control group mice [(61.66±10.53)% vs (49.31±4.3)%, respectly. P>0.05]. We have compared three different strategies for separation of CD34⁺ cells from mouse bone marrow and analyzed purity and the cell viability for each individual strategy. Our results indicate that all three strategies (positive selection, negative selection and negative selection combined with positive selection) can efficiently enrich CD34⁺ cells and there were significant difference before and after purification [(2.29±0.52)% vs (54.79±5.32)%, (1.62±0.72)% vs (7.67±1.46)%, (1.62 ±0.72)% vs (70.09±4.02)%, respectively. P<0.05]. Similar results for purity were obtained using positive selection and negative selection combined with positive selection [(54.79±5.32)% vs (70.09±4.02)%, respectively. P>0.05]. The cell viability was not notably impaired in all three selection strategies [(94.22±2.95)% vs (99.00±1.32)%, (96.75±0.96)% vs (99.00±0.82)%, (93.75±1.71)% vs (99.00±0.82)%, respectively. P>0.05].Conclusion: Relative highly purified CD34⁺ cells can be obtained from mouse bone marrow using magnetic cell-sorting system (MACS) with positive selection or negative selection combined with positive selection which does not impair the viability of the derived cells.Objective: To investigate the effect of the eotaxin/CCR3 axis in eosinophilia and the migration and differentiation of bone marrow CD34⁺ progenitors in allergic mice and to assess the effect of anti-CCR3 monoclonal antibody.Methods: C57BL/6 mice were randomly divided into four groups: NS/NS group, OVA/OVA group, OVA/CCR3mAb group, OVA/IgG group. In the OVA/OVA group, mice were sensitized and challenged with chicken OVA. In NS/NS group, mice were sensitized and challenged with saline by the same methods. In the OVA/CCR3mAb group, mice were injected intraperitoneally with anti-CCR3 monoclonal antibody before each aerosol OVA challenge. In the OVA /ns-IgG group, mice were administered the same amounts of rat ns-IgG by same way. Eosinophil, CD34⁺ progenitor and CD34⁺CCR3⁺ cell populations in bronchial alveolar lavage fluid (BALF), blood and bone marrow were determined after antigen challenge. The airway responsiveness was conducted by barometric whole-body plethysmography. Chemokine receptor expression was analyzed by using flow cytometry. In vitro chemotaxis and colony-forming assay were also performed.Results: Airway hyperreactivity, mucus overproduction and eosinophil infilitrtion in the lung parenchyma were observed in OVA/OVA group after antigen challenge. The number of eosinophil, CD34⁺ progenitor and CD34⁺CCR3⁺ cell populations in BALF were remarkablely increased after antigen challenge in OVA/OVA group compared with NS/NS group. The correlation analysis indicated that the number of eosinophil in BALF was closely correlated with CD34⁺/CCR3⁺ cells population in bone marrow. However, there was no significant correlation between the number of eosinophil in BALF and the number of CD34⁺ progenitors in bone marrow. In vivo administrated with anti-CCR3 monoclonal antibodysignificantly decreased airway hyperreactivity and mucus overproduction, and suppressed the increased eosinophils, CD34⁺ cells and CD34⁺/CCR3⁺cells population in BALF in OVA-challenged mice compared with ns-IgG-treated mice. In vitro anti-CCR3 monoclonal antibody reduced the increased Eo-CFU when addition eotaxin in IL-5 induced culture and the migratory response of bone marrow CD34⁺ progenitor cells to eotaxin.Conclusion: The expression of CCR3 on bone marrow CD34⁺ progenitors was up-regulated after allergen challenge. The eotaxin/CCR3 axis plays an important role in eosinophilia and the migration and differentiation of bone marrow progenitors in allergic inflammation. CCR3mAb can inhibit the eosinophilia and the migration and differentiation of bone marrow progenitors in allergic inflammation context. Therapeutic blockade of CCR3 may be a novel strategy for the treatment of asthma.Part3The eotaxin/CCR3 axis and the migration and differentiation of bone marrow CD34⁺ progenitors in allergic Mice: Effect ofGlucocorticoidsObjective: To investigate the effect of Glucocorticoids in eosinophilia and the migration and the differentiation of CD34⁺ progenitors in allergic mice.Methods: C57BL/6 mice were randomly divided into three groups: NS/NS group, OVA/OVA group, OVA/DXM group. In the OVA/OVA group, mice were sensitized and challenged with chicken OVA. In NS/NS group, mice were sensitized and challenged with normal saline by the same methods. In the OVA/DXM group, mice were treated with an intraperitoneal injection of dexamethasone lhr before each aerosol OVA challenge. Eosinophil, CD34⁺ progenitor and CD34⁺CCR3⁺ progenitor population were determined after antigen challenge. The airway responsiveness wasconducted by barometric whole-body plethysmography. CC chemokine receptor 3 (CCR3) was analyzed by using flow cytometry and real-time PCR. The technique of in situ hybridization was used to detect mouse IL-5Ra mRNA and Immunocyto-chemistry was used to detect mouse CD34 antigen on mouse bone marrow cells. In vitro chemotaxis and colony-forming assay were also performed.Results: Administration with dexamethasone in vivo (OVA/DXM group) effectively ablated eosinophils, CD34⁺ progenitor and CD34⁺CCR3⁺ cell populations in BALF and significantly reduced lung eosinophila induced by allergen challenge compared with the OVA/OVA group. In addition, administration with dexamethasone in vivo significantly reduced airway hyperreactivity and mucus overproduction induced by allergen challenge. The expression of CCR3 and IL-5Rα mRNA on bone marrow CD34⁺ progenitor cells were significantly up-regulated at 48 hr after the last allergen exposure in OVA/OVA group compared with NS/NS group. However, we did not detect any significant up-regulation of expression of CCR3 or IL-5Rα mRNA on bone marrow CD34⁺ progenitor cells from OVA/DXM group mice after allergen exposure. In vitro, CD34⁺ progenitor cells from OVA/DXM group mice had a lower chemotactic response to eotaxin than those from OVA/OVA group mice. In cultures of NAMNC isolated from the OVA/OVA group mice with IL-5, the addition of eotaxin further increased the number of Eos-CFU. However, the similar story did not happened in cultures of NAMNC isolated from the OVA/DXM group miceConclusion: Administration with dexamethasone in vivo can inhibit eosinophilia, reduce airway hyperreactivity and mucus overproduction induced by allergen challenge. In vivo dexamethasone can inhibit the up-regulate of the expression of CCR3 and IL-5Ra mRNA on bone marrow progenitor induced by allergen challenge. Dexamethasone partly via regulation of the eotaxin/CCR3 axis, exert indirect suppressive effects on the migration and differentiation of eosinophil progenitors in allergic inflammation context.SummaryIn the present study, we first assessed the efficiency of magnetic activated cell sorting (MACS) separation in purification and enrichment of CD34⁺ cells from asthmatic mouse bone marrow with three selection strategies. And then, we investigated the effect of the eotaxin/CCR3 axis in the migration and the differentiation of bone marrow CD34⁺ progenitors in allergic mice and assessed the effect of anti-CCR3 monoclonal antibody and dexamethasone in this process. We obtained some conclusion as follwing :1. Relative highly purified CD34⁺ cells can be obtained from mouse bone marrow using magnetic cell-sorting system (MACS) with positive selection or negative selection combined with positive selection which does not impair the viability of the derived cells.2. The expression of CCR3 on bone marrow CD34⁺ progenitors was up-regulated after allergen challenge.3. The eotaxin/CCR3 axis plays an important role in eosinophilia and the migration and differentiation of bone marrow progenitors in allergic inflammation.4. CCR3mAb can inhibit the eosinophilia and the migration and differentiation of bone marrow progenitors in allergic inflammation context. Therapeutic blockade of CCR3 may be a novel strategy for the treatment of asthma.5. In vivo dexamethasone can inhibit eosinophilia, reduce airway hyperreactivity and mucus overproduction induced by allergen challenge.6. Administration with dexamethasone in vivo may inhibit the up-regulate of the expression of CCR3 and IL-5Ra mRNA on bone marrow progenitor induced by allergen challenge.7. Dexamethasone partly via regulation of the eotaxin/CCR3 axis, exert indirect suppressive effects on the migration and differentiation of eosinophil progenitors in allergic inflammation context.