The Role Of IL-1β In The Differentiation Of Neutrophil And Eosinophil In Bone Marrow

Asthma refers to a chronic airway inflammatory diseases characterized by airway inflammation, mucin overproduction,airway hyperresponsiveness, fibroblasts and epithelial cells metaplasia, smooth muscle cells hyperplasia and airway remodeling, which is associated with many kinds of cells and inflammatory factor, such as eosinophils, neutrophils, lymphocytes, mast cells, cytokines and chemokines milieu. Asthma is conventionally considered to be a T helper2(Th2)-related eosinophil inflammation,which is sensitive to glucocorticoid therapy. However, patients with refractory asthma which are difficult to control and the main airway inflammatory cell is neutrophil, not eosinophil. They are resistant to high doses of inhaled corticosteroid, and even some patients experience asthma exacerbations or irreversible airway obstruction. The refractory asthma is also called neutrophilic asthma or severe asthma. Since eosinophil and neutrophil play important roles in the pathogenesis and clinical manifestation of asthma, it would bring momentous significance to effective asthma therapeutics if we take measures to block eosinophilic and neutrophilic inflammation in airway.IL-1β belongs to IL-1family of cytokines, acts as an important pro-inflammatory factor in many diseases. Overproduction of IL-1β is thought to be responsible for asthma, especially the severe asthma, contributing to the neutrophil and eosinophil infiltration, while the mechanism is little known. Evidence from recent reports consistently implicated IL-1β could be used as a biomarker to distinguish asthma from chronic obstructive pulmonary disease. According to several studies, IL-1β also acts on bone marrow stem cells for differentiation of the myeloid series of progenitor cells though it is unclear. Therefore, we hypothesize that IL-1β could function directly on neutrophil and eosinophil differentiation from hematopoietic stem and progenitor cells. Moreover, it will provide theoretical foundation for exploring the pathogenesis of asthma, and bring the new insight into the preventation and therapy of asthma.Objective:To explore the effect of IL-1β in neutrophil and eosinophil differentiation and the underlying molecular mechanism, bringing the new insight into the theoretical foundation of asthma.Method:Healthy wild type(WT) C57BL/6mice and IL-1receptor knock out(IL-1R-/-) mice were divided into four groups randomly:WT-NS group; WT-LPS group; IL-1R-/--NS group and IL-1R-/--LPS group. In the WT-LPS and IL-1R-/-LPS group, mice were instilled of lmg/kg LPS intratracheally(i.t., lmg/kg),while in the WT-NS group and IL-1R-/--NS group, mice were received intratracheal administration of NS instead of LPS.24hours after the intervention,mice were sacrificed. Total number of BALF and BM cells were counted, and furthermore the percentage of neutrophils in bronchoalveolar fluid (BALF), peripheral blood(PB) and bone marrow(BM) were accessed. BM cells were isolated and labeled with neutrophil surface Abs (Gr-1, CD11b). Neutrophil were determined by staining anti-Gr-1and anti-CD llb+cells mAbs which are consist of three distinct double-positive populations in corresponding differentiation status. After sorting to high purity, the primitive Neutrophil (Pro/mye) were considered as Gr-int CD11bint cells, including promyelocytes and myelocytes, Gr-1hiCDllb10cells represented an intermediate stage of neutrophil maturation (metamyelocytes and band forms, imNeut), whereas mature Neutrophils (hypersegmented neutrophil, mNeut) were detected as Gr-lhiCDllbhi cells. In preparation for bone marrow transplantation (BMT), recipient female WT mice were treated with BU(i.p., mg/kg) for continuous four days and then treated with CTX(i.p., mg/kg) for two days.48h after CTX administrated, BM cells were obtained from the femurs of donor male WT mice and IL-1R-/-mice,2-4×107bone marrow cells were needed to transplanted into recipients via tail vein infusion. By day21after BMT, peripheral lymphohematopoietic reconstitution of all cell lineages could recover to be normal and the recipients were divided into the following groups:WT-WT-NS-group: recipients received WT donor bone marrow cells were adminstrated NS(i.t); WT-WT-LPS-group:recipients received WT donors’bone marrow cells were adminstrated LPS(i.t.,1mg/kg); IL-1R-/--WT-NS-group:recipients received IL-1R-/-donors’ bone marrow cells were adminstrated NS(i.t). IL-1R-/--WT-LPS-group:recipients received IL-1R-/-donors’ bone marrow cells were adminstrated LPS(i.t.,1mg/kg).24hours after LPS intervention, the mice were disposed as the same as previously described. In vitro methylcellulose semi-solid culture were used to observe the influence of IL-1β in neutrophil colony forming, standing for the ability of cell differentiation traditionally. Liquid culture of mouse bone marrow-derived neutrophils (BM-Neut) were also performed to assess the ability of neutrophil differentiation、apoptosis and phagocytic ability with neutrophil surface Abs (Gr-1,CD11b), Annexin V/PI and E.coli phagocytosis Assay kit. All the FACS data were collected and analyzed by using a Beckman flow cytometry. The mRNA expression of several key transcription factors, which played the critical role in neutrophil differentiation were detected by Q-PCR such as G-CSFR, C/EBPe, PU.l and C/EBPa. In vitro experiment to detect the role of IL-1β in eosinophil differentiation:Methylcellulose colony forming assays and liquid culture of mouse bone marrow-derived eosinophils (BM-Eos) were performed to assess the ability of eosinophil differentiation; In liquid culture, bone marrow cells were detected by labeling with eosinophil surface Abs, eosinophil were determined as SiglecF+cells and BrdU were used to investigate the proliferation and apoptosis of eosinophil; The mRNA of important transcription factor GATA-1and regulatory protein MBP-1were detected by Q-PCR.Results:An LPS-induced neutrophilic inflammation model was successfully established in which IL-1β level had been highlighted to be increased significantly according to several literatures.Comparing the IL-1R-/--LPS group with WT-LPS group, the absolute number of neutrophils in BALF had a tendency to decrease, though without statistical significancy(P=0.0571), the percentage of neutrophils in peripheral blood were attenuated (P<0.05), bone marrow Pro/mye didn’t change evidently (P>0.05), while imNeuts was increased obviously(P<0.05), and mNeuts were reduced significantly(P<0.001). In contrast to WT-WT-LPS group, the airway neutrophilic inflammation in IL-1R-/--WT-LPS group in contrast to WT-WT-LPS group also had a tendency to relieved though without statistical significance (P=0.0503) in bone marrow transplantation test, and PB neutrophils was lessened obviously(P<0.05). Bone marrow Pro/mye and imNeuts both didn’t change evidently (P>0.05), while mNeuts was decreased remarkably(P<0.001). It demonstrated that pre-treatment of IL-1β in coordination with G-CSF significantly promoted the differentiation(P<0.001) and phagocytosis (P<0.05) in vitro liquid culture, without inhibiting the apoptosis of neutrophils(P>0.05), whereas IL-1β alone was insufficient.It also observed that robust neutrophil colonies were formed under the intervention of IL-1β and G-CSF in vitro semi-solid culture. Moreover, in WT-LPS group, mRNA expression of G-CSFR, C/EBPe, PU.1and C/EBPa, the critical transcription factors during neutrophil differentiation, were up-regulated in comparison with WT-NS group(P<0.05), while there was no difference between IL-1R-/--LPS group and IL-1R-/--NS group, this phenomenon did not occur(P>0.05). In vitro eosinophil colony forming unit assay and liquid culture demonstrated that pre-treatment of IL-1β didn’t bring significant change to the differentiation and proliferation of eosiniphil (P>0.05). The important transcription factor and calculator, GATA-1and MBP-1, were not obviously changed(P>0.05). However, it Is found that the apoptotic cell population was diminished with IL-1β intervention in comparison with IL-5administrated alone.Conclusion:IL-1β significantly promoted the G-CSF mediated differentiation of neutrophils, while IL-1β alone did not have that effect. The underlying mechanisms may be due to the up-regulation of the expression of G-CSFR, C/EBPe, PU.1and C/EBPa. In the meanwhile, IL-1β could promote the phagocytosis of neutrophils, while don’t have the function in inhibiting the apoptosis of neutrophils. In addition, IL-1βdid not have an effect on the differentiation of eosinophils, however, IL-1βcould inhibit the apoptosis of eosinophils.