The Study of the Influence of IL5RA Variants on Chronic Obstructive Pulmonary Disease.

Chronic obstructive pulmonary disease (COPD) is a complex disease, and its pathogenesis is influenced by genetic factors. This study aimed to evaluate the role of IL5RA genetic variation in the risk of COPD. In this study, 498 patients with COPD and 498 normal controls were recruited. Subsequently, five SNPs (rs3804795, rs2290610, rs13097407, rs334782, and rs3856850) in the IL5RA gene were genotyped. Logistic analysis examined the association of five single nucleotide polymorphisms (SNPs) in IL5RA with the risk of COPD under various genetic models. Furthermore, the association between IL5RA and susceptibility to COPD was comprehensively analyzed with stratification based on age, sex, smoking, and alcohol consumption. Our study showed that IL5RA rs13097407 reduced susceptibility to COPD (OR = 0.43, p < 0.001, p (FDR)< 0.001). On the other hand, rs3856850 was associated with an increased risk of COPD (OR = 1.71, p = 0.002, p (FDR) = 0.002). Interestingly, the effect of IL5RA SNPs on susceptibility to COPD was found to be influenced by factors such as sex and smoking. IL5RA gene variants were significantly associated with susceptibility to COPD.

IL5RA as an immunogenic cell death-related predictor in progression and therapeutic response of multiple myeloma.

Previous studies have shown the potential of immunogenic cell death-related modalities in myeloma. The significance of IL5RA in myeloma and immunogenic cell death remains unknown. We analyzed IL5RA expression, the gene expression profile, and secretory protein genes related to IL5RA level using GEO data. Immunogenic cell death subgroup classification was performed using the ConsensusClusterPlus and pheatmap R package. Enrichment analyses were based on GO/KEGG analysis. After IL5RA-shRNA transfection in myeloma cells, cell proliferation, apoptosis, and drug sensitivity were detected. P < 0.05 was considered statistically significant. IL5RA was upregulated in myeloma and progressed smoldering myeloma. We observed enrichment in pathways such as the PI3K-Akt signaling pathway, and Natural killer cell mediated cytotoxicity in the high-IL5RA group. IL5RA was also closely associated with secretory protein genes such as CST6. We observed the enrichment of cellular apoptosis and hippo signaling pathway on differential genes in the immunogenic cell death cluster. Furthermore, IL5RA was associated with immune infiltration, immunogenic cell death-related genes, immune-checkpoint-related genes, and m6A in myeloma. In vitro and in vivo experiments showed the involvement of IL5RA in apoptosis, proliferation, and drug resistance of myeloma cells. IL5RA shows the potential to be an immunogenic cell death-related predictor for myeloma.

Transcriptional Regulation of the Human IL5RA Gene through Alternative Promoter Usage during Eosinophil Development.

Regulation of the IL-5 receptor alpha (IL5RA) gene is complicated, with two known promoters (P1 and P2) driving transcription, and two known isoforms (transmembrane and soluble) dichotomously affecting the signaling potential of the protein products. Here, we sought to determine the patterns of P1 and P2 promoter usage and transcription factor occupancy during primary human eosinophil development from CD34+ hematopoietic stem cell progenitors. We found that during eosinophilopoiesis, both promoters were active but subject to distinct temporal regulation, coincident with combinatorial interactions of transcription factors, including GATA-1, PU.1, and C/EBP family members. P1 displayed a relatively constant level of activity throughout eosinophil development, while P2 activity peaked early and waned thereafter. The soluble IL-5Rα mRNA peaked early and showed the greatest magnitude fold-induction, while the signaling-competent transmembrane isoform peaked moderately. Two human eosinophilic cell lines whose relative use of P1 and P2 were similar to eosinophils differentiated in culture were used to functionally test putative transcription factor binding sites. Transcription factor occupancy was then validated in primary cultures by ChIP. We conclude that IL-5-dependent generation of eosinophils from CD34+ precursors involves complex and dynamic activity including both promoters, several interacting transcription factors, and both signaling and antagonistic protein products.

House Dust Mite Induces Bone Marrow IL-33-Responsive ILC2s and TH Cells.

Type 2 innate lymphoid cells (ILC2s) and their adaptive counterpart type 2 T helper (TH2) cells respond to interleukin-33 (IL-33) by producing IL-5, which is a crucial cytokine for eosinophil development in the bone marrow. The aim of this study was to determine if bone marrow ILC2s, TH cells, and eosinophils are locally regulated by IL-33 in terms of number and activation upon exposure to the common aeroallergen house dust mite (HDM). Mice that were sensitized and challenged with HDM by intranasal exposures induced eosinophil development in the bone marrow with an initial increase of IL5Rα+ eosinophil progenitors, following elevated numbers of mature eosinophils and the induction of airway eosinophilia. Bone marrow ILC2s, TH2, and eosinophils all responded to HDM challenge by increased IL-33 receptor (ST2) expression. However, only ILC2s, but not TH cells, revealed increased ST2 expression at the onset of eosinophil development, which significantly correlated with the number of eosinophil progenitors. In summary, our findings suggest that airway allergen challenges with HDM activates IL-33-responsive ILC2s, TH cells, and eosinophils locally in the bone marrow. Targeting the IL-33/ST2 axis in allergic diseases including asthma may be beneficial by decreasing eosinophil production in the bone marrow.

B-cells with a FasL expressing regulatory phenotype are induced following successful anti-tuberculosis treatment.

INTRODUCTION: Studies show that B-cells, in addition to producing antibodies and antigen-presentation, are able to produce cytokines as well. These include regulatory cytokines such as IL-10 by regulatory B-cells. Furthermore, a rare regulatory subset of B-cells have the potential to express FasL, which is a death-inducing ligand. This subset of B-cells have a positive role during autoimmune disease, but has not yet been studied during tuberculosis. These FasL-expressing B-cells are induced by bacterial LPS and CpG, thus we hypothesized that this phenotype might be induced during tuberculosis as well. METHODS: B-cells from participants with TB (at diagnosis and during treatment) and controls were collected, and analyzed by means of real-time PCR and flow cytometry. In addition to this, BAL was collected from TB participants as well and analyzed by means of MAGPix (multi-cytokine) technology. RESULTS: Gene expression analysis show that FASL transcript levels increase by the end of treatment. Similarly, phenotypic analysis show that there is a higher frequency of FasL-expressing B-cells by the end of treatment. CONCLUSION: Collectively, these results indicate that these FasL-expressing B-cells are being induced during anti-TB treatment, and thus may play a positive role. Further studies are required to elucidate this.

IL-5 blocks apoptosis and tau hyperphosphorylation induced by Aß25-35 peptide in PC12 cells.

The primary features of Alzheimer's disease (AD) are extracellular amyloid plaques consisting mainly of deposits of amyloid ß (Aß) peptides and intracellular neurofibrillary tangles (NFTs). Sets of evidence suggest that interleukin-5 (IL-5) is involved in the pathogenesis of AD. Herein, we investigated the protective role of IL-5 in PC12 cells, to provide new insights into understanding this disease. Western blot was employed to assess the protein levels of Bax and phospho-tau as well as phospho-JAK2; MTT assay was performed to decipher cell viability. Treatment of IL-5 decreased Aß25-35-induced tau phosphorylation and apoptosis, effects blunted by JAK2 inhibition. IL-5 prevents Aß25-35-evoked tau protein hyperphosphorylation and apoptosis through JAK2 signaling.

Successful TB treatment induces B-cells expressing FASL and IL5RA mRNA.

Activated B-cells increase T-cell behaviour during autoimmune disease and other infections by means of cytokine production and antigen-presentation. Functional studies in experimental autoimmune encephalomyelitis (EAE) indicate that B-cell deficiencies, and a lack of IL10 and IL35 leads to a poor prognosis. We hypothesised that B-cells play a role during tuberculosis. We evaluated B-cell mRNA expression using real-time PCR from healthy community controls, individuals with other lung diseases and newly diagnosed untreated pulmonary TB patients at three different time points (diagnosis, month 2 and 6 of treatment).We show that FASLG, IL5RA, CD38 and IL4 expression was lower in B-cells from TB cases compared to healthy controls. The changes in expression levels of CD38 may be due to a reduced activation of B-cells from TB cases at diagnosis. By month 2 of treatment, there was a significant increase in the expression of APRIL and IL5RA in TB cases. Furthermore, after 6 months of treatment, APRIL, FASLG, IL5RA and CD19 were upregulated in B-cells from TB cases. The increase in the expression of APRIL and CD19 suggests that there may be restored activation of B-cells following anti-TB treatment. The upregulation of FASLG and IL5RA indicates that B-cells expressing regulatory genes may play an important role in the protective immunity against M.tb infection. Our results show that increased activation of B-cells is present following successful TB treatment, and that the expression of FASLG and IL5RA could potentially be utilised as a signature to monitor treatment response.

IL5RA polymorphisms, smoking and eczema in Japanese women: the Kyushu Okinawa Maternal and Child Health Study.

The present case-control study examined the relationship between IL5RA SNPs and eczema in young adult Japanese women. Cases and control subjects were selected from pregnant women who participated in the baseline survey of the Kyushu Okinawa Maternal and Child Health Study, which is an ongoing prebirth cohort study. Cases comprised 188 women with eczema in the previous 12 months as defined according to the criteria of the International Study of Asthma and Allergies in Childhood (ISAAC), regardless of the presence of a doctor's diagnosis of atopic eczema. Control subjects comprised 1130 women without eczema as defined according to the ISAAC criteria who also had not been diagnosed with atopic eczema by a doctor. Compared with the AA genotype of IL5RA SNP rs17881144, the AT genotype, but not the TT genotype, was significantly associated with a decreased risk of eczema. The ATTAGA haplotype and the GTAGCA haplotype of rs17882210, rs3804797, rs334809, rs9831572, rs6771148 and rs17881144 were significantly associated with an increased risk of eczema. In contrast, the GCTGCA haplotype was significantly related to a decreased risk of eczema. Multiplicative interactions between IL5RA SNPs rs334809 and rs17881144 and smoking with respect to eczema were marginally significant (P = 0.07 and 0.07, respectively). This is the first study to show significant associations between IL5RA SNP rs17881144, the ATTAGA haplotype, the GTAGCA haplotype, and the GCTGCA haplotype and eczema. Smoking may modify the relationships between SNPs rs334809 and rs17881144 and eczema.

Case-control study of rhinoconjunctivitis associated with IL5RA polymorphisms in Japanese women: the Kyushu Okinawa Maternal and Child Health Study.

BACKGROUND: Epidemiological research on the relationship between single nucleotide polymorphisms (SNPs) in the IL5RA gene and allergic disorders is limited. We examined the relationship between IL5RA SNPs and risk of rhinoconjunctivitis in young adult Japanese women. METHODS: Included were 393 women who met the criteria of the International Study of Asthma and Allergies in Childhood (ISAAC) for rhinoconjunctivitis. Controls were 767 women without rhinoconjunctivitis according to the ISAAC criteria who had not been diagnosed with allergic rhinitis by a doctor. Adjustment was made for age, region of residence, presence of older siblings, smoking, and education. RESULTS: Compared with the CC genotype of SNP rs6771148, the CG genotype, but not the GG genotype, was significantly associated with a reduced risk of rhinoconjunctivitis: the adjusted OR for the CG genotype was 0.76 (95% CI: 0.58-0.99). No evident associations were found between SNPs rs17882210, rs3804797, rs334809, rs9831572, or rs17881144 and rhinoconjunctivitis. The ACTAGA haplotype of rs17882210, rs3804797, rs334809, rs9831572, rs6771148, and rs17881144 was significantly inversely associated with rhinoconjunctivitis (crude OR=0.58, 95% CI: 0.37-0.88) while the GTAGCA haplotype was significantly positively related to rhinoconjunctivitis (crude OR=1.74, 95% CI: 1.14-2.65). No significant interactions affecting rhinoconjunctivitis were observed between any of the six SNPs and smoking. CONCLUSION: This is the first study to show significant associations between IL5RA SNP rs6771148, the ACTAGA haplotype, and the GTAGCA haplotype and the risk of rhinoconjunctivitis. We did not find evidence for interactions affecting rhinoconjunctivitis between any of the IL5RA SNPs and smoking.

A genetic effect of IL-5 receptor α polymorphism in patients with aspirin-exacerbated respiratory disease.

Persistent eosinophil activation in both the upper and lower airway mucosa is a central feature of aspirin-exacerbated respiratory disease (AERD). Eosinophil activation and survival are profoundly influenced by interleukin 5 (IL-5) and its receptor, IL-5R. In patients susceptible to allergic disorders, IL-5 receptor α (IL5RA) polymorphisms have been reported; however, an association with AERD remains unclear. We hypothesize that IL5RA polymorphisms may contribute to eosinophil activation in AERD patients. We recruited 139 AERD patients, 171 aspirin-tolerant asthma patients and 160 normal controls. IL5RA polymorphisms (-5993G>A, -5567C>G and -5091G>A) were genotyped and functional activity of polymorphism was assessed by luciferase reporter assay and electrophoretic mobility shift assay (EMSA). There was no significant difference in the genotype frequency of the three polymorphisms among the three groups. AERD patients carrying the AA genotype at -5993G>A had a significantly higher presence of serum-specific immunoglobulin E (IgE) to staphylococcal enterotoxin A (P=0.008) than those with the GG/GA genotype. In vitro, the -5993A allele had a higher promoter activity compared with the -5993G allele in human mast cell (HMC-1; P=0.030) and human promyelocytic leukemia (HL-60; P=0.013) cells. In EMSA, a -5993A probe produced a specific shifted band than the -5993G had. These findings suggest that a functional polymorphism in IL5RA may contribute to eosinophil and mast cell activation along with specific IgE responses to staphylococcal enterotoxin A in AERD patients.

Purification of functional eosinophils from human bone marrow.

Eosinophils are granulocytic leukocytes that are best known for their involvement in host immune defense and pathologic states. More recently, they have also been shown to play a role in regulation of murine plasma cell homeostasis in the bone marrow, which prompted our investigation of human bone marrow eosinophils. However, effective methods to isolate eosinophils from human bone marrow thereby allowing comparisons with circulating eosinophils have not yet been described. Herein we describe the development of a novel, cost effective protocol for the purification of eosinophils from human bone marrow that allows us to obtain bone marrow eosinophils of near 100% purity after an 8-day culture system. Furthermore, we demonstrate that bone marrow eosinophils have characteristics similar to blood eosinophils, including the expression of IL-5Rα, the presence of eosinophil-specific granules, and similar activation kinetics upon phorbol myristate acetate and high-dose IL-5 stimulation. While migratory responses toward the chemokine CXCL12 differed between purified bone marrow and freshly isolated blood eosinophils, migratory responses were similar upon comparison of bone marrow eosinophils with blood eosinophils cultured ex vivo for 8 days prior to assay. Interestingly, a concurrent upregulation of CXCR4 expression was not observed in these cultured blood eosinophils. Taken together, we have overcome the existing challenges to the study of bone marrow eosinophils through our novel strategy for cell purification and have thus enabled future investigations of these cells and their role(s) in human health and disease.

IL-5-induced migration via ERK1/2-mediated MMP-9 expression by inducing activation of NF-κB in HT1376 cells.

Interleukin-5 (IL-5) plays an important role in the growth and differentiation of human B cells and eosinophils. However, little is known about the effect of IL-5 on cancer cells. In this study, we investigated the molecular mechanisms involved in the IL-5-induced migration of HT1376 bladder cancer cells. Our results indicated that IL-5 significantly enhanced migration and MMP-9 expression in HT1376 cells. We also found that IL-5 induces transcriptional activation of the binding of NF-κB and AP-1, which are two important nuclear transcription factors that are linked to MMP-9 expression in HT1376 cells. In subsequent experiments, we found activation of ERK1/2 in IL-5-treated HT1376 cells. To examine the involvement of the ERK1/2 signaling pathway on IL-5-induced cell responses, we pretreated HT1376 cells with the ERK1/2 inhibitor U0126 followed by IL-5 treatment. The results showed that U0126 treatment inhibited migration of IL-5-treated HT1376 cells. Moreover, IL-5-stimulated MMP-9 expression was suppressed by the addition of U0126. Inhibition of ERK1/2 function consistently rescued transcriptional activity of NF-κB, without altering AP-1 activation, in IL-5-treated cells. Finally, inhibition of the IL-5-specific receptor IL-5Rα by small interfering RNA (siRNA) suppressed migration, ERK1/2 activation, MMP-9 expression and binding activation of NF-κB in IL-5-treated HT1376 cells. The results of the present study indicate that the IL-28A/IL-28AR1 dyad induces cell migration through ERK1/2-mediated expression of MMP-9 by binding activation of NF-κB in bladder cancer cells. In conclusion, these novel findings indicate that binding of IL-5 to IL-5Rα plays a critical role in MMP-9 expression, which may be involved in the migration of bladder cancer.

Molecular cloning of chicken interleukin-5 receptor α-chain and analysis of its binding specificity.

Interaction between interleukin (IL)-5 and its receptor (IL-5R) is important for the regulation of immunity against worm infections, allergic reactions and B cell response in mammals. In this study, we identified a full-length cDNA encoding chicken IL-5R α-chain (chIL-5Rα). The deduced amino acid sequence showed 41-43% identity to mammalian homologues. It has four well-conserved cysteines and a WSXWS motif in the extracellular region, and a PPXP motif in the cytoplasmic region. Quantitative RT-PCR analysis revealed that chIL-5Rα mRNA expression was markedly high in bone marrow and relatively high in spleen and lung. Recombinant proteins of soluble chIL-5Rα and cytokines (artificially produced chIL-5 (achIL-5) and another IL-5-like molecule KK34) were expressed by 293F cells to examine the cytokine-receptor interactions. Interaction assay using a Biacore biosensor showed that chIL-5Rα has the capability to bind with monomeric achIL-5, but not with KK34. In conclusion, chicken has an IL-5Rα homologue but KK34 does not complement the IL-5/IL-5R system.

Fractionation of mature eosinophils in GATA-reporter transgenic mice.

Eosinophils contribute to the pathophysiology of allergic and infectious diseases, albeit their molecular functions remain unknown. Mature eosinophils are identified by their unique morphology and staining characteristics. However, it is difficult to fractionate living eosinophils by flow cytometry because these granulocytes express multiple cell surface markers that are shared by other cells of hematopoietic or non-hematopoietic origin. In this study, we describe a flow cytometry-based method to enumerate and fractionate eosinophils by developmental stages. To fractionate these cell types, we used transgenic mouse lines that express fluorescent proteins under control of the Gata1 gene hematopoietic regulatory region (Gata1-HRD), which is exclusively active in Gata1-expressing hematopoietic cells, including eosinophils. As expected, mature eosinophils were highly enriched in the fluorescent reporter-expressing subfraction of bone marrow myeloid cells that were negatively selected by using multiple antibodies against B220, CD4, CD8, Ter119, c-Kit and CD71. Cytochemical analyses of flow-sorted cells identified the cells in this fraction as eosinophils harboring eosinophilic granules. Additionally, expression of eosinophil-specific genes, for instance eosinophil enzymes and the IL-5 receptor alpha gene, were specifically detected in this fraction. The expression of these eosinophil-specific genes increased as the cells differentiated. This method for enrichment of bone marrow eosinophils is applicable to fractionation of eosinophils and bronchoalveolar lavage fluid from transgenic mice with atopic asthma. Thus, both pathological and developmental stages of eosinophils are efficiently fractionated by this flow cytometry-based method using Gata1-HRD transgenic reporter mice. This study, therefore, proposes a useful means to study the experimental allergic and inflammatory systems.

Differential expression of the interleukin 5 receptor alpha isoforms in blood and tissue eosinophils of nasal polyp patients.

BACKGROUND: Given the key role of interleukin-5 (IL-5) in eosinophil function, we investigated the regulated expression of the membrane-anchored (TM-IL-5Ralpha) isoform, or a secreted (SOL IL-5Ralpha) isoform, on both protein and transcript level in vitro and in vivo. METHODS: A real-time PCR, FACS and ELISA were established to determine IL-5Ralpha isoform expression in peripheral blood and nasal tissue from control subjects and nasal polyp (NP) patients with or without asthma. Human peripheral blood eosinophils were incubated with IL-5 and were analyzed for SOL-IL-5Ralpha and TM-IL-5Ralpha mRNA and protein levels in comparison with CD-69 expression. RESULTS: SOL-IL-5Ralpha and TM-IL-5Ralpha mRNA and protein expression was significantly increased in NP vs controls. In polyp tissue, SOL-IL-5Ralpha expression correlated to disease severity and eosinophils counts, whereas TM-IL-5Ralpha levels were inversely correlated to eosinophils counts and SOL-IL-5Ralpha expression. FACS analysis revealed increased CD-69 and decreased TM-IL-5Ralpha expression in NP tissue eosinophils vs blood eosinophils. Incubation of blood eosinophils with IL-5 caused up-regulation of CD-69 and down-regulation of TM-IL-5Ralpha after 2 and 24 h. CONCLUSION: The expression of SOL-IL-5Ralpha and TM-IL-5Ralpha differs according to the eosinophil activation state and localization in the body (blood vs tissue) and may therefore be involved in the fine-tuning of the eosinophil homeostasis. Exposure of eosinophils to IL-5 reduces their responsiveness to IL-5 by regulated expression of the IL-5Ralpha isoforms. Since, TM-IL-5Ralpha is down-regulated and SOL-IL-5Ralpha (antagonistic) is upregulated in NP tissue, our findings are important to understand the clinical trials with anti-IL-5 in humans.

IL5RA and TNFRSF6B gene variants are associated with sporadic IgA nephropathy.

Familial clustering and genome-wide linkage scans strongly support a genetic susceptibility to familial IgA nephropathy (IgAN), but genetic factors that predispose to sporadic IgAN are unknown. A high-throughput single nucleotide polymorphism (SNP) association study was conducted using a customized Illumina BeadChip in 732 white patients with biopsy-proven IgAN and 503 control subjects from Canada, France, and Finland. Approximately 93% of 1536 SNPs on the array were tag SNPs from Phase I+II of the HapMap with a minor allele frequency > or =5%, designed to capture the common variants of genes within the critical interval of IGAN1 on chromosome 6q22 and 69 biologic candidate genes for IgAN. SNPs of suggestive or significant association were identified by using logistic regression to adjust for age, gender, study site, and population stratification. Despite using a dense marker set that covered an average interval of 6.5 kb between SNPs, there was no strong and consistent association signal within the IGAN1 critical interval. Among the biologic candidate genes examined, two significant association signals were found at IL5RA and TNFRSF6B, the latter being particularly interesting because this gene encodes a decoy receptor for a TNF family ligand that causes IgAN in mice when overexpressed. Pending replication, these data suggest that variants of IL5RA and TNFRSF6B may predispose to sporadic IgAN.

The severity of FIP1L1-PDGFRA-positive chronic eosinophilic leukaemia is associated with polymorphic variation at the IL5RA locus.

We have investigated the hypothesis that constitutional genetic variation in IL-5 signalling may be associated with the development or severity of FIP1L1-PDGFRA-positive chronic eosinophilic leukaemia (CEL) in humans. We genotyped six single-nucleotide polymorphisms (SNP) within or close to the IL5RA or IL5 genes in 82 patients with FIP1L1-PDGFRA-positive CEL plus, as controls, healthy individuals (n=100), patients with FIP1L1-PDGFRA-negative eosinophilia (n=100) or patients with chronic myeloid leukaemia (CML) (n=100). We found no association between SNP allele frequency between FIP1L1-PDGFRA-positive and control cases. However, for FIP1L1-PDGFRA cases, we found an association between the genotype at rs4054760, an SNP in the 5'-UTR of IL5RA and peripheral blood eosinophil count (P=0.026) as well as the presence or absence of tissue infiltration (P=0.032). Although these associations fell below the level of significance once corrected for multiple testing, no such association was seen in FIP1L1-PDGFRA-negative cases and no difference in allele frequencies for rs4054760 was seen in control populations across Europe. Furthermore, in an analysis of 112 patients with CML, IL5RA expression was strongly related to rs4054760 genotype (P<0.001). These data suggest that the variations in IL5RA expression are linked to constitutional IL5RA genotype and severity of FIP1L1-PDGFRA disease.

Effect of inhaled interleukin-5 on eosinophil progenitors in the bronchi and bone marrow of asthmatic and non-asthmatic volunteers.

BACKGROUND: Asthma is characterized by increases in mature eosinophils and their progenitors within the bronchus and bone marrow. IL-5 plays a key role in eosinophil development in the bone marrow and at the site of allergic inflammation. We therefore studied the effects of nebulized IL-5 on eosinophils, their progenitors and in situ haemopoiesis within the airway and bone marrow. METHODS: Nine atopic asthmatics and 10 non-atopic non-asthmatic control volunteers inhaled 10 microg of IL-5 or placebo via a nebulizer in a double-blind, randomized, cross-over study. Bronchoscopy, bone marrow aspiration and peripheral blood sampling were performed 24 h after nebulization. Four weeks later, volunteers inhaled the alternative solution and underwent a repeat bronchoscopy and bone marrow aspiration. RESULTS: Inhalation of IL-5 significantly decreased CD34(+)/IL-5Ralpha mRNA(+) cells within the bronchial mucosa and the percentage of CD34(+) cells that were CCR3(+) within the bone marrow of atopic asthmatic, but not control, volunteers. Inhalation of IL-5 also induced a significant increase in bronchial mucosal eosinophils in the non-atopic non-asthmatic control volunteers, but not in the asthmatics. IL-5 had no effect on spirometry or airways hyper-reactivity in either group. CONCLUSIONS: Inhaled IL-5 modulated eosinophil progenitor numbers in both the airways and bone marrow of asthmatics and induced local eosinophilia in non-asthmatics.

A preliminary study towards downregulation of murine bone marrow eosinophilopoiesis mediated by small molecule inhibition of interleukin-5 receptor alpha gene in vitro.

BACKGROUND: Bone marrow eosinophilopoiesis induced by IL-5 makes a major contribution to eosinophilic airway inflammation in asthma. Bone marrow CD(34)(+) cells expressing IL-5Ralpha may be eosinophil progenitors. However, research on the effect of blocking IL-5Ralpha expression on bone marrow eosinophilopoiesis has seldom been reported. OBJECTIVE: To explore the effect of inhibiting IL-5Ralpha expression with IL-5Ralpha short hairpin RNA-expressing vector on murine bone marrow eosinophilopoiesisin vitro. METHODS: We constructed 4 kinds of plasmid vectors that could express small molecule inhibition, short hairpin RNA, which targeted IL-5Ralpha (P-IL-5Ralpha), and selected an effective one by transfecting B lymphoma cells in vitro. We also constructed an adenovirus vector which was inserted into an effective template sequence (Ad-IL-5Ralpha). The bone marrow cells were obtained from healthy Balb/c mice, and cultured and transfected by Ad-IL-5Ralpha in vitro. The expression of IL-5Ralpha and the count of newly produced eosinophils were detected in the cultured bone marrow cells. RESULTS: We found that P-IL-5Ralpha-3 targeted at the sequence of CAG CTG CCT GGT TCG TCT T markedly suppressed the IL-5Ralpha expression in the B lymphoma cellsin vitro. Ad-IL-5Ralpha could suppress the IL-5Ralpha expression of murine bone marrow cellsin vitro and it could also significantly decrease the IL-5-induced eosinophilia in the cultured bone marrow cells. CONCLUSION: These results indicate that the blocking of IL-5Ralpha expression by small molecule inhibition can help to effectively decrease murine bone marrow eosinophilopoiesis, and that bone marrow may be used as a critical target organ in the diseases involved in eosinophilia, such as asthma.

Effect of interleukin-5 receptor-alpha short hairpin RNA-expressing vector on bone marrow eosinophilopoiesis in asthmatic mice.

Bone marrow eosinophilopoiesis induced by interleukin (IL)-5 is a major contributor to eosinophilic airway inflammation in asthma. However,research on the use of IL-5 receptor alpha (IL-5Ralpha) as the target has seldom been reported. This study was undertaken to explore the effects of inhibition of IL-5Ralpha expression through an IL-5Ralpha short hairpin RNA-expressing vector on bone marrow eosinophilopoiesis and airway inflammation in an asthmatic mouse model. An effective plasmid vector was selected that could express short hairpin RNA targeted at IL-5Ralpha (P-IL-5Ralpha). An adenovirus vector (Ad) was then constructed that was inserted in an effective template sequence (Ad-IL-5Ralpha). An animal model of asthma was established by sensitizing and challenging Balb/c mice with ovalbumin. Animals were treated intravenously with Ad-IL-5Ra and changes in bone marrow eosinophilopoiesis and airway inflammation were detected in asthmatic mice. Investigators found that P-IL-5Ra-3 targeted at the sequence of CAG CTG CCT GGT TCG TCT T markedly suppressed IL-5Ralpha expression in B lymphoma cells in vitro. In addition, Ad-IL-5Ralpha could suppress IL-5Ralpha expression in murine bone marrow cells in vitro and in vivo, and it could significantly decrease IL-5-induced eosinophilia in cultured bone marrow cells. Additional studies indicated that intravenously injected Ad-IL-5Ralpha not only selectively reduced the number of eosinophils in the bone marrow, peripheral blood, and bronchoalveolar lavage fluid, it also relieved airway inflammation in asthmatic mice. Results reported here show that blocking of IL-5Ralpha expression through RNA interference can enhance effective treatment of asthma, and that bone marrow can be used as a key targeted organ in the treatment of asthmatic mice.