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Specific therapeutics are not available for acute lung injury (ALI) induced by ricin toxin (RT). Inhibiting the host immune response in the course of pulmonary ricinosis is hypothesized to be of benefit and can be achieved by impairing granulocyte-macrophage colony-stimulating factor (GM-CSF) signaling, thereby reducing the pro-inflammatory response to exogenous foreign body invasion. However, it is unknown whether mice with impaired GM-CSF signaling can survive after RT inhalation. To test this, colony stimulating factor 2 receptor alpha (Csf2ra) knockout (KO) mice that lack GM-CSF signaling and wild-type (WT) mice models of intratracheal exposure to a lethal dose (2× LD50) of RT were established. Survival was greater in Csf2ra KO mice 21 days after RT inhalation compared with WT mice. Highly coexpressed genes that probably attenuated the pro-inflammatory response in the lung of Csf2ra KO mice were identified. Bioinformatics analysis revealed that transcriptome changes involved mostly inflammation-related genes after RT exposure in both Csf2ra KO mice and WT mice. However, the activity levels of pro-inflammatory pathways, such as the TNF signaling pathway and NF-kB signaling pathway, in Csf2ra KO mice were significantly decreased and the degree of neutrophil chemotaxis and recruitment inhibited after RT-exposure relative to WT mice. RT-qPCR and flow cytometry validated results of RNA-Seq analysis. This work provides potential avenues for host-directed therapeutic applications that can mitigate the severity of ALI-induced by RT. [ABSTRACT FROM AUTHOR]

Hereditary pulmonary alveolar proteinosis (hPAP) is a rare disorder caused by recessive mutations in GM-CSF receptor subunit a/b genes (CSF2RA/CSF2RB, respectively) characterized by impaired GM-CSF-dependent surfactant clearance by alveolar macrophages (AMs) resulting in alveolar surfactant accumulation and hypoxemic respiratory failure. Because hPAP is caused by CSF2RA mutations in most patients, we created an animal model of hPAP caused by Csf2ra gene disruption (Csf2ra-/- mice) and evaluated the effects on AMs and lungs. Macrophages from Csf2ra-/- mice were unable to bind and clear GM-CSF, did not exhibit GM-CSF signaling, and had functional defects in phagocytosis, cholesterol clearance, and surfactant clearance. Csf2ra-/- mice developed a time-dependent, progressive lung disease similar to hPAP in children caused by CSF2RA mutations with respect to the clinical, physiological, histopathological, biochemical abnormalities, biomarkers of PAP lung disease, and clinical course. In contrast, Csf2ra+/- mice had functionally normal AMs and no lung disease. Pulmonary macrophage transplantation (PMT) without myeloablation resulted in long-term engraftment, restoration of GM-CSF responsiveness to AMs, and a safe and durable treatment effect that lasted for the duration of the experiment (6 mo). Results demonstrate that homozygous (but not heterozygous) Csf2ra gene ablation caused hPAP identical to hPAP in children with CSF2RA mutations, identified AMs as the cellular site of hPAP pathogenesis in Csf2ra-/- mice, and have implications for preclinical studies supporting the translation of PMT as therapy of hPAP in humans. [ABSTRACT FROM AUTHOR]

Dendritic cells (DCs) are key antigen-presenting cells that prime naive T cells and initiate adaptive immunity. Although the genetic deficiency and transgenic overexpression of granulocyte macrophage-colony stimulating factor (GM-CSF) signaling were reported to influence the homeostasis of DCs, the in vivo development of DC subsets following injection of GM-CSF has not been analyzed in detail. Among the treatment of mice with different hematopoietic cytokines, only GM-CSF generates a distinct subset of XCR1-33D1- DCs which make up the majority of DCs in the spleen after three daily injections. These GM-CSF-induced DCs (GMiDCs) are distinguished from classical DCs (cDCs) in the spleen by their expression of CD115 and CD301b and by their superior ability to present blood-borne antigen and thus to stimulate CD4+ T cells. Unlike cDCs in the spleen, GMiDCs are exceptionally effective to polarize and expand T helper type 2 (Th2) cells and able to induce allergic sensitization in response to blood-borne antigen. Single-cell RNA sequencing analysis and adoptive cell transfer assay reveal the sequential differentiation of classical monocytes into pre-GMiDCs and GMiDCs. Interestingly, mixed bone marrow chimeric mice of Csf2rb+/+ and Csf2rb-/- demonstrate that the generation of GMiDCs necessitates the cis expression of GM-CSF receptor. Besides the spleen, GMiDCs are generated in the CCR7-independent resident DCs of the LNs and in some peripheral tissues with GM-CSF treatment. Also, small but significant numbers of GMiDCs are generated in the spleen and other tissues during chronic allergic inflammation. Collectively, our present study identifies a splenic subset of CD115hiCD301b+ GMiDCs that possess a strong capacity to promote Th2 polarization and allergic sensitization against blood-borne antigen. [ABSTRACT FROM AUTHOR]

Objectives: As the prognosis of relapsed/refractory (R/R) acute myeloid leukaemia (AML) remains poor, novel treatment strategies are urgently needed. Clinical trials have shown that chimeric antigen receptor (CAR)‐T cells for AML are more challenging than those targeting CD19 in B‐cell malignancies. We recently developed piggyBac‐modified ligand‐based CAR‐T cells that target CD116/CD131 complexes, also known as the GM‐CSF receptor (GMR), for the treatment of juvenile myelomonocytic leukaemia. This study therefore aimed to develop a novel therapeutic method for R/R AML using GMR CAR‐T cells. Methods: To further improve the efficacy of the original GMR CAR‐T cells, we have developed novel GMR CAR vectors incorporating a mutated GM‐CSF for the antigen‐binding domain and G4S spacer. All GMR CAR‐T cells were generated using a piggyBac‐based gene transfer system. The anti‐tumor effect of GMR CAR‐T cells was tested in mouse AML xenograft models. Results: Nearly 80% of the AML cells predominant in myelomonocytic leukaemia were found to express CD116. GMR CAR‐T cells exhibited potent cytotoxic activities against CD116+ AML cells in vitro. Furthermore, GMR CAR‐T cells incorporating a G4S spacer significantly improved long‐term in vitro and in vivo anti‐tumor effects. By employing a mutated GM‐CSF at residue 21 (E21K), the anti‐tumor effects of GMR CAR‐T cells were also improved especially in long‐term in vitro settings. Although GMR CAR‐T cells exerted cytotoxic effects on normal monocytes, their lethality on normal neutrophils, T cells, B cells and NK cells was minimal. Conclusions: GMR CAR‐T cell therapy represents a promising strategy for CD116+ R/R AML. [ABSTRACT FROM AUTHOR]

Abstract Objectives As the prognosis of relapsed/refractory (R/R) acute myeloid leukaemia (AML) remains poor, novel treatment strategies are urgently needed. Clinical trials have shown that chimeric antigen receptor (CAR)‐T cells for AML are more challenging than those targeting CD19 in B‐cell malignancies. We recently developed piggyBac‐modified ligand‐based CAR‐T cells that target CD116/CD131 complexes, also known as the GM‐CSF receptor (GMR), for the treatment of juvenile myelomonocytic leukaemia. This study therefore aimed to develop a novel therapeutic method for R/R AML using GMR CAR‐T cells. Methods To further improve the efficacy of the original GMR CAR‐T cells, we have developed novel GMR CAR vectors incorporating a mutated GM‐CSF for the antigen‐binding domain and G4S spacer. All GMR CAR‐T cells were generated using a piggyBac‐based gene transfer system. The anti‐tumor effect of GMR CAR‐T cells was tested in mouse AML xenograft models. Results Nearly 80% of the AML cells predominant in myelomonocytic leukaemia were found to express CD116. GMR CAR‐T cells exhibited potent cytotoxic activities against CD116+ AML cells in vitro. Furthermore, GMR CAR‐T cells incorporating a G4S spacer significantly improved long‐term in vitro and in vivo anti‐tumor effects. By employing a mutated GM‐CSF at residue 21 (E21K), the anti‐tumor effects of GMR CAR‐T cells were also improved especially in long‐term in vitro settings. Although GMR CAR‐T cells exerted cytotoxic effects on normal monocytes, their lethality on normal neutrophils, T cells, B cells and NK cells was minimal. Conclusions GMR CAR‐T cell therapy represents a promising strategy for CD116+ R/R AML.

Abstract Background Juvenile myelomonocytic leukemia (JMML) is a fatal, myelodysplastic/myeloproliferative neoplasm of early childhood. Patients with JMML have mutually exclusive genetic abnormalities in granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor (GMR, CD116) signaling pathway. Allogeneic hematopoietic stem cell transplantation is currently the only curative treatment option for JMML; however, disease recurrence is a major cause of treatment failure. We investigated adoptive immunotherapy using GMR-targeted chimeric antigen receptor (CAR) for JMML. Methods We constructed a novel CAR capable of binding to GMR via its ligand, GM-CSF, and generated piggyBac transposon-based GMR CAR-modified T cells from three healthy donors and two patients with JMML. We further evaluated the anti-proliferative potential of GMR CAR T cells on leukemic CD34+ cells from six patients with JMML (two NRAS mutations, three PTPN11 mutations, and one monosomy 7), and normal CD34+ cells. Results GMR CAR T cells from healthy donors suppressed the cytokine-dependent growth of MO7e cells, but not the growth of K562 and Daudi cells. Co-culture of healthy GMR CAR T cells with CD34+ cells of five patients with JMML at effector to target ratios of 1:1 and 1:4 for 2 days significantly decreased total colony growth, regardless of genetic abnormality. Furthermore, GMR CAR T cells from a non-transplanted patient and a transplanted patient inhibited the proliferation of respective JMML CD34+ cells at onset to a degree comparable to healthy GMR CAR T cells. Seven-day co-culture of GMR CAR T cells resulted in a marked suppression of JMML CD34+ cell proliferation, particularly CD34+CD38− cell proliferation stimulated with stem cell factor and thrombopoietin on AGM-S3 cells. Meanwhile, GMR CAR T cells exerted no effects on normal CD34+ cell colony growth. Conclusions Ligand-based GMR CAR T cells may have anti-proliferative effects on stem and progenitor cells in JMML.

Abstract: The damaging and degenerative effects in autoimmune diseases such as rheumatoid arthritis, multiple sclerosis and Crohn's disease often manifests as the formation of lesions that feature a high local concentration of granulocyte–macrophage colony‐stimulating factor (GM‐CSF). GM‐CSF along with other pro‐inflammatory factors form a positive feedback loop that ultimately perpetuate the lesions. Hence, to engineer chemotaxis to GM‐CSF, we created a new chimeric GM‐CSF receptor alpha subunit (GMRchi) that was coupled with a previously engineered Ca2+‐activated RhoA. When these proteins were expressed in mammalian cells, it allowed migration to chemical and cellular sources of GM‐CSF. As a possible therapeutic intervention, we further implemented the mechanism of cell–cell membrane fusion and subsequent death. Since the microenvironment of lesions is more than just GM‐CSF secretion, the further ability to recognize a combination of other features such as tissue markers will be needed for greater specificity. Nonetheless, this work represents a first step to enable cell‐based therapy of autoimmune lesions. [ABSTRACT FROM AUTHOR]

Background Eosinophilic esophagitis (EoE) is a chronic antigen-mediated clinicopathologic disease of the esophagus characterized by an eosinophil-predominant inflammatory infiltrate. A clinical hallmark is extensive tissue remodeling including basal zone hyperplasia, fibrosis, and angiogenesis. However, the cellular mechanisms responsible for these processes are not fully defined. We hypothesized that targeting granulocyte-macrophage colony-stimulating factor ( GM- CSF; an agonist cytokine linked with eosinophil survival and activation) would be protective in a preclinical model of EoE. Methods Eosinophilic esophagitis-like esophageal inflammation was induced in the L2- IL5OXA EoE mouse model, and GM- CSF production was assessed by m RNA and protein analyses. Granulocyte-macrophage colony-stimulating factor-receptor-alpha expression patterns were examined by flow cytometric and immunofluorescence analysis. L2- IL5OXA EoE mice were treated with anti- GM- CSF neutralizing antibody or isotype control and assessed for histopathological indices of eosinophilia, epithelial hyperplasia, and angiogenesis by immunohistochemistry and RT- PCR. Results Significantly increased levels of esophageal GM- CSF expression was detected in the L2- IL5OXA mouse EoE model during active inflammation. Granulocyte-macrophage colony-stimulating factor-receptor-alpha was predominantly expressed on esophageal eosinophils during EoE, in addition to select cells within the lamina propria. Anti- GM- CSF neutralization in L2- IL5OXA EoE mice resulted in a significant diminution of epithelial eosinophilia in addition to basal cell hyperplasia and vascular remodeling. This treatment response was independent of effects on esophageal eosinophil maturation or activation. Conclusion Granulocyte-macrophage colony-stimulating factor is a potential therapeutic target to reduce esophageal eosinophilia and remodeling. [ABSTRACT FROM AUTHOR]

Background: Pulmonary alveolar proteinosis (PAP) is a rare lung disease characterized by surfactant accumulation, and is caused by disruption of granulocyte/macrophage colony-stimulating factor (GM-CSF) signaling. Abnormalities in CSF2 receptor alpha (CSF2RA) were reported to cause pediatric hereditary PAP. We report here the first case of CSF2RA-mutated, elderly-onset hereditary (h) PAP.Case Presentation: The patient developed dyspnea on exertion, and was diagnosed with PAP at the age of 77 years, based on findings from chest computed tomography scan and bronchoalveolar lavage. She tested negative for GM-CSF autoantibodies, with no underlying disease. Her serum GM-CSF level was elevated (91.3 pg/mL), indicating GM-CSF signaling impairment and genetic defects in the GM-CSF receptor. GM-CSF-stimulated phosphorylation in signal transducer and activator of transcription 5 (STAT5) was not observed, and GM-CSF-Rα expression was defective in her blood cells. Genetic screening revealed a homozygous, single-base C > T mutation at nt 508-a nonsense mutation that yields a stop codon (Q170X)-in exon 7 of CSF2RA. High-resolution analysis of single nucleotide polymorphism array confirmed a 22.8-Mb loss of heterozygosity region in Xp22.33p22.11, encompassing the CSF2RA gene. She was successfully treated with whole lung lavage (WLL), which reduced the serum levels of interleukin (IL)-2, IL-5, and IL-17, although IL-3 and M-CSF levels remained high.Conclusions: This is the first known report of elderly-onset hPAP associated with a CSF2RA mutation, which caused defective GM-CSF-Rα expression and impaired signaling. The analyses of serum cytokine levels during WLL suggested that GM-CSF signaling might be compensated by other signaling pathways, leading to elderly-onset PAP. [ABSTRACT FROM AUTHOR]

Background: Juvenile myelomonocytic leukemia (JMML) is a fatal, myelodysplastic/myeloproliferative neoplasm of early childhood. Patients with JMML have mutually exclusive genetic abnormalities in granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor (GMR, CD116) signaling pathway. Allogeneic hematopoietic stem cell transplantation is currently the only curative treatment option for JMML; however, disease recurrence is a major cause of treatment failure. We investigated adoptive immunotherapy using GMR-targeted chimeric antigen receptor (CAR) for JMML. Methods: We constructed a novel CAR capable of binding to GMR via its ligand, GM-CSF, and generated piggyBac transposon-based GMR CAR-modified T cells from three healthy donors and two patients with JMML. We further evaluated the anti-proliferative potential of GMR CAR T cells on leukemic CD34+ cells from six patients with JMML (two NRAS mutations, three PTPN11 mutations, and one monosomy 7), and normal CD34+ cells. Results: GMR CAR T cells from healthy donors suppressed the cytokine-dependent growth of MO7e cells, but not the growth of K562 and Daudi cells. Co-culture of healthy GMR CAR T cells with CD34+ cells of five patients with JMML at effector to target ratios of 1:1 and 1:4 for 2 days significantly decreased total colony growth, regardless of genetic abnormality. Furthermore, GMR CAR T cells from a non-transplanted patient and a transplanted patient inhibited the proliferation of respective JMML CD34+ cells at onset to a degree comparable to healthy GMR CAR T cells. Seven-day co-culture of GMR CAR T cells resulted in a marked suppression of JMML CD34+ cell proliferation, particularly CD34+CD38- cell proliferation stimulated with stem cell factor and thrombopoietin on AGM-S3 cells. Meanwhile, GMR CAR T cells exerted no effects on normal CD34+ cell colony growth. Conclusions: Ligand-based GMR CAR T cells may have anti-proliferative effects on stem and progenitor cells in JMML. [ABSTRACT FROM AUTHOR]

Highlights: [•] GM-CSF is a pro-inflammatory cytokine. [•] GM-CSF is a key factor in Th17 driven autoimmune inflammatory conditions. [•] GM-CSF is a therapeutic target for autoimmune inflammatory diseases. [ABSTRACT FROM AUTHOR]

Granulocyte-monocyte colony-stimulating factor (GM-CSF) is a member of the hematopoietic growth factor family, promoting proliferation and differentiation of hematopoietic progenitor cells of the myeloid lineage. In recent years, GM-CSF has also proved to be an important neurotrophic factor in the central nervous system (CNS) via binding to the GM-CSF receptor (GM-CSF R). Furthermore, studies on rodent CNS revealed a wide distribution of both the major binding α-subunit of the GM-CSF R (GM-CSF Rα) and its ligand. Since respective data on the expression pattern of these two molecules are still lacking, the present study has been designed to systematically analyze the protein expression of GM-CSF and GM-CSF Rα in the human brain, with particular emphasis on their regulation in Alzheimer's disease (AD). One major finding is that both GM-CSF and GM-CSF Rα were ubiquitously but not uniformly expressed in neurons throughout the CNS. Protein expression of GM-CSF and GM-CSF Rα was not restricted to neurons but also detectable in astrocytes, ependymal cells and choroid plexus cells. Interestingly, distribution and intensity of immunohistochemical staining for GM-CSF did not differ among AD brains and age-matched controls. Concerning GM-CSF Rα, a marked reduction of protein expression was predominantly detected in the hippocampus although a slight reduction was also found in various cortical regions, thalamic nuclei and some brainstem nuclei. Since the hippocampus is one of the target regions of neurodegenerative changes in AD, reduction of GM-CSF Rα, with consecutive downregulation of GM-CSF signaling, may contribute to in the progressive course of neurodegeneration in AD. [ABSTRACT FROM AUTHOR]

Abstract: We studied the expression of granulocyte–macrophage colony-stimulating factor (GM-CSF) and its receptors (GM-CSF.R) in 20 human brain gliomas with different tumor gradings and demonstrated constitutive high levels of both mRNA gene expression and protein production exclusively in the highest-grade tumors (WHO, III–IV grade). Five astrocytic cell lines were isolated in vitro from glioma cells, which had selectively adhered to plates pre-coated with rhGM-CSF. These cells were tumorigenic when xenografted to athymic mice, and produced GM-CSF constitutively in culture. Two lines, particularly lines AS1 and PG1, each from a patient with glioblastoma multiforme, constitutively over-expressed both GM-CSF and GM-CSF.R genes and secreted into their culture media biologically active GM-CSF. Different clones of the AS1 line, isolated after subsequent passages in vitro and then transplanted to athymic mice, demonstrated higher tumorigenic capacity with increasing passages in vivo. Cell proliferation was stimulated by rhGM-CSF in late-stage malignant clones, whereas apoptosis occurred at high frequency in the presence of blocking anti-GM-CSF antibodies. In contrast, rhGM-CSF did not induce any apparent effect in early-stage clones expressing neither GM-CSF nor GM-CSF.R. The addition of rhGM-CSF or rhIL-1β, to cultures induced the overproduction of both GM-CSF and its receptors and increased gene activation for several functional proteins (e.g. NGF, VEGF, VEGF.R1, G-CSF, MHC-II), indicating that these cells may undergo dynamic changes in response to environmental stimuli. These findings thus revealed: (1) that the co-expression of both autocrine GM-CSF and GM-CSF.R correlates with the advanced tumor stage; (2) that an important contribution of GM-CSF in malignant glioma cells is the prevention of apoptosis. These results imply that GM-CSF has an effective role in the evolution and pathogenesis of gliomas. [Copyright &y& Elsevier]

Background Disruption of granulocyte/macrophage colony-stimulating factor (GM-CSF) signalling causes pulmonary alveolar proteinosis (PAP). Rarely, genetic defects in neonatal or infant-onset PAP have been identified in . However, no report has clearly identified any function-associated genetic defect in . Methods and results The patient was diagnosed with PAP at the age of 36 and developed respiratory failure. She was negative for GM-CSF autoantibody and had no underlying disease. Signalling and genetic defects in GM-CSF receptor were screened. GM-CSF-stimulated STAT5 phosphorylation was not observed and GM-CSF-Rβc expression was defective in the patient's blood cells. Genetic screening revealed a homozygous, single-base deletion at nt 631 in exon 6 of on chromosome 22, which caused reductions in GM-CSF dependent signalling and function. Both parents, who were second cousins, showed no pulmonary symptoms, and had normal GM-CSF-signalling, but had a allele with the identical deletion, indicating that the mutant allele may give rise to PAP in an autosomal recessive manner. Conclusions This is the first report identifying a genetic defect in that causes deficiency of GM-CSF-Rβc expression and impaired signalling downstream. These results suggested that GM-CSF signalling was compensated by other signalling pathways, leading to adult-onset PAP. [ABSTRACT FROM PUBLISHER]

Granulocyte macrophage colony-stimulating factor (GM-CSF) is a cytokine that promotes myeloid cell development and maturation, and dendritic cell differentiation and survival in vitro. Growing evidence supports the notion that GM-CSF has a major role in some inflammatory and autoimmune reactions and in the host's response to pulmonary infection, but few studies have addressed its functions and importance in the GI tract. Recent studies demonstrated that administration of GM-CSF can result in clinical improvement in patients with Crohn's disease. Mice deficient in GM-CSF (GM-CSF-l-) developed more severe intestinal and systemic infection after an enteric infection, and more severe colitis in response to enteric exposure to dextran sodium sulfate. Both the severity of infection and colitis were largely prevented by GM-CSF administration. Such studies indicate that GM-CSF has an important role in the regulation of intestinal immune and inflammatory responses. [ABSTRACT FROM AUTHOR]

Pulmonary alveolar proteinosis (PAP) is a rare disease of the lung characterized by the accumulation of surfactant-derived lipoproteins within pulmonary alveolar macrophages and alveoli, resulting in respiratory insufficiency and increased infections. The disease is caused by a disruption in surfactant catabolism by alveolar macrophages due to loss of functional granulocyte-macrophage colony-stimulating factor (GM-CSF) signaling. The underlying molecular mechanisms causing deficiencies in GM-CSF signaling are as follows: 1) high levels of neutralizing GM-CSF autoantibodies observed in autoimmune PAP; 2) mutations in CSF2RA, the gene encoding the α chain of the GM-CSF receptor, observed in hereditary PAP; and 3) reduced numbers and function of alveolar macrophages as a result of other clinical diseases seen in secondary PAP. Recent studies investigating the biology of GM-CSF have revealed that not only does this cytokine have an indispensable role in lung physiology, but it is also a critical regulator of innate immunity and lung host defense. [ABSTRACT FROM AUTHOR]

Abstract: A girl with congenital dysgranulopoietic neutropenia (CDN) and her non-neutropenic mother with aphthae (A) were investigated. Apoptosis in lymphocytes and granulocytes of both patients (mother A+) were documented by high annexin and electron microscopic morphology. CD11b/CD18 of the daughter''s granulocytes ranged between low to normal while that of the mother changed between very low to high levels through A(−) to A(+) periods. In both patients, CD11b/CD18 on lymphocytes were high; GM-CSF receptor was negative; CD4−/CD8− lymphocytes were high and the leukocytes which showed abnormal cell cycle were stained by senescence associated β-galactosidase. We think that increased apoptosis and rapid cell senescence of leukocytes underlies the pathophysiology of CDN. [Copyright &y& Elsevier]

Immunotoxins, which are composed of both the cell targeting and the cell killing moieties are the new approach for targeted therapy of human disease .In all immunotoxins that GM-CSF has been used as cell targeting; only cell lines expressing high affinity receptor have been used for cytotoxicity studies. In the present study, various cell lines expressing high and low affinity receptors were used for assessment of the cytotoxic effect of hybrid chimeric protein. The expression of GM-CSF receptor (GM-CSFR) was quantified by real-time RT- PCR. The cell lines K562 and THP1 expressing high affinity receptor and MC-7, PC-3 and DU145 expressing low affinity receptor were used for this study. The chimeric hybrid protein was found to be toxic for various cell lines used in this investigation and cytotoxicity was more effective in cell lines bearing high affinity receptors. Overall, our results showed that the recombinant hybrid protein could have wide range of application on various cancer cell lines even cells bearing low affinity receptors for GM-CSF. [ABSTRACT FROM AUTHOR]

Abstract: Background: In remodelling ventricles, the progression of heart failure is associated with structural changes involving the extra-cellular matrix (ECM) and the cytoskeleton of cardiomyocytes, associated with fibrosis, cellular damage and death. The role of some cytokines and haematopoietic growth factors in the mechanism of both damage and regeneration of cardiac tissue during acute myocardial infarction has been demonstrated. Following heart damage, the development of scarred tissue was considered to be the only outcome, since myocytes were considered to be terminally differentiated cells. However, recent studies in animal models and adult human hearts have shown that myocytes can proliferate under the modulation of several factors. Aims: To assess Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF) receptor expression in healthy and diseased human hearts, and to evaluate the possible role of GM-CSF and its receptor in the regeneration of cardiac tissue in chronic cardiomyopathy. Methods and results: GM-CSFR expression in human cardiac tissue from explanted hearts of ten patients with end-stage heart failure and in cardiac biopsies from eight normal human hearts was studied by immunohistochemistry, and cellular and molecular biology assays. Our results demonstrated an increase in GM-CSFR in cardiomyocytes from end-stage heart failure tissues as compared to normal control tissues. Conclusions: We hypothesize that GM-CSF plays a role in apoptotic and/or ECM deposition processes as well as in cytoskeleton modification in the myocardium. [Copyright &y& Elsevier]

Granulocyte Macrophage Colony Stimulating Factor (GM-CSF) has been inducted as a mediator of inflammation in rheumatoid arthritis. Methotrexate combination therapy forms an important component of the treatment regimen in rheumatoid arthritis. The present study was undertaken to evaluate the influence of Methotrexate-Hydroxychloroquine (MTX-HCQ) combination and Sulfsalazine- Hydroxychloroquine (SSZ-HCQ) combination on the expression GM-CSFR in neutrophils isolated from synovial fluids. 15 cases of confirmed rheumatoid arthritis patients who presented at the hospital for surgical correction of joint deformities were selected for the study. Neutrophils isolated from the synovial fluids were used as the source of the receptor for quantitation on an enzyme immunoassay (EIA). The EIA was developed and standardized in our laboratory for quantification of the GM-CSF R. The findings are suggestive of the fact that the administration of MTX-HCQ combination has positive influence on the expression of the GM-CSF R on neutrophils as against SSZ-HCQ combination. The physiological basis of this increase needs further investigation. [ABSTRACT FROM AUTHOR]

The hematopoietic factor and inflammatory cytokine GM-CSF is involved in PNS and CNS injury and disease, and in macrophage and microglia function regulation. We presently document that injury to PNS axons induces in vivo production of GM-CSF-inhibitor and GM-CSF-augmenter activities. GM-CSF-inhibitor activity was detected in extract and conditioned medium (CM) of injured PNS but not in extract of intact PNS, and was removed from CM by GM-CSF affinity chromatography, suggesting it is carried by a secreted GM-CSF binding molecule. CM further displayed GM-CSF-augmenter activity along with GM-CSF-inhibitor activity but at contrasting concentrations; augmentation at lowest and inhibition at highest. GM-CSF activity is thus regulated during Wallerian degeneration (WD); augmenter activity characterizes the onset and inhibitor activity the later stages of WD. [Copyright &y& Elsevier]

Summary Investigation of differentially expressed genes in eosinophils of patients with allergic diseases such as atopic dermatitis (AD) will provide important information for elucidating possible mechanisms of pathology. To identify novel genes that are expressed in AD, we compared gene expression in samples of peripheral blood eosinophils from AD patients and healthy volunteers. RNA was extracted from peripheral blood eosinophils. The expression of various genes, such as those for cytokine receptors, eosinophil activation marker, platelet activating factor (PAF) receptor, eosinophil-specific granular proteins and apoptosis-related genes, was confirmed using real-time reverse transcription–polymerase chain reaction (RT-PCR). Peripheral blood eosinophils of healthy volunteers were also isolated and stimulated for introduction of various cytokines. RNA was extracted and gene expression was monitored. Several genes, such as those for cytokine receptors (granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor α and β chain and interleukin (IL)-3 receptor α chain), CD44 and PAF receptor were expressed at significantly higher levels in AD patients than in healthy volunteers. In addition, the anti-apoptotic genes, bcl-2 and bcl-xL, were expressed at increased levels in AD patients. No single gene expression correlated with clinical markers, such as eosinophil count or IgE levels. Expression of GM-CSF receptor β chain and IL-3 receptor α chain in isolated blood eosinophils of healthy volunteers was stimulated by IL-5, IL-4, interferon (IFN)-γ and GM-CSF. Expression of bcl-2 and bcl-xL was also increased after stimulation with IL-5, IL-4 or IFN-γ . The in vitro enhancement of cytokine-stimulated gene expression correlated well with the enhancement observed in clinical samples of eosinophils, suggesting that cytokines may affect gene expression in vivo in eosinophils of patients with AD. [ABSTRACT FROM AUTHOR]

The receptors for human interleukin-3 (hIL-3R) and granulocyte–macrophage colony-stimulating factor (hGM-CSFR) consist of an α subunit, specific for each cytokine, and a β subunit, common to IL-3, GM-CSF, and IL-5. We cloned genomic DNA covering 1.5 kb of the 5′ flanking region of the hIL-3Rα gene and identified multiple transcription start sites by 5′-RACE and primer extension analyses. By use of transient transfection experiments, two regions (nt −363 to −331 and −106 to −92) of the hIL-3Rα promoter appeared to have significant transcription-enhancing activities. Electrophoresis mobility shift assays revealed the binding of Sp1 and unidentified proteins to these regions. Deletion of a putative PU.1 binding site did not affect the promoter activity. We then analyzed 2.5 kb of the hGM-CSFR α gene and found the proximal PU.1 binding site to be important for transcription-enhancing activity, as previously reported. These results suggest that different transcriptional activation mechanisms are employed for the transcriptional regulation of hIL-3 and hGM-CSF receptor α genes. [Copyright &y& Elsevier]

In vitro exposure of murine bone marrow cells to increasing concentrations of zidovudine (AZT, 0.1–50 μM) had a concentration dependent suppressive effect on the growth of granulocyte-monocyte colony forming unit (CFU-GM) derived colonies. In our previous published study, the mechanism of AZT-induced suppression of erythroid colony forming unit (CFU-E) derived colonies was linked to a decrease in erythropoitin receptor (Epo-R) gene expression. In this study, we have observed that AZT exposure also induced a concentration dependent suppressive effect (35–90%) on GM-CSF receptor type alpha (GM-CSFRα) gene expression. The suppression of GM-CSFRα mRNA expression was specific, since AZT caused a much lower decrease (15–22%) on the IL-3 receptor type alpha (IL-3Rα) message level, and had an insignificant effect on glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and c-myc message levels. Erythropoietin (Epo) therapy has been used for reversal of AZT induced erythroid toxicity. Exposure to increasing concentrations (10–500 U/ml) of GM-CSF was unable to override the suppressive effect of AZT on CFU-GM derived colonies, however, treatment in combination with IL-3 (10–250 U/ml) ameliorated the suppressive effects of AZT on CFU-GM and on GM-CSFRα and IL-3Rα gene expression. These findings suggest a mechanism via which AZT may suppress granulocyte-monocyte specific differentiation in murine bone marrow cells. These data also suggest that a combination of GM-CSF and IL-3 may be a superior therapeutic intervention for AZT-induced neutropenia. [Copyright &y& Elsevier]

Background: The expression and function of cytokine receptors on peripheral blood eosinophils (PBE) from healthy and asthmatic children are poorly characterized. Methods: The PBE count and expression of IL-5 receptor (R) and GM-CSFR positive PBE was analyzed in nonsteroid-treated asthmatic children (n = 13), budesonide-treated asthmatic children (n = 24) and healthy children (n = 16) by flow cytometry. Alterations in intracellular EG2-epitope expression were used to measure the in vitro responsiveness of PBE to recombinant IL-5 and GM-CSF. Results: The PBE count was increased (P < 0.05) in both asthmatic groups, independent of treatment, as compared to healthy children. The IL-5R expression on PBE, as well as the in vitro responsiveness of PBE to recombinant IL-5, was reduced (P < 0.05), in budesonide-treated asthmatic children compared to nonsteroid-treated asthmatic children and healthy children. The proportion of GM-CSFR positive PBE and in vitro responsiveness of PBE to recombinant GM-CSF were not different between the groups. In vitro treatment with budesonide did not down-regulate the proportion of IL-5R positive PBE. Conclusions: Budesonide-treatment of asthmatic children induces a selectively reduced IL-5R expression on PBE, concomitant with a reduced in vitro responsiveness of PBE to IL-5. We suggest that this budesonide-related down-regulation of the IL-5R might be a mechanism by which steroid treatment inhibits the action of IL-5 on eosinophil accumulation and activation in vivo . Background: The expression and function of cytokine receptors on peripheral blood eosinophils (PBE) from healthy and asthmatic children are poorly characterized. Methods: The PBE count and expression of IL-5 receptor (R) and GM-CSFR positive PBE was analyzed in nonsteroid-treated asthmatic children (n = 13), budesonide-treated asthmatic children (n = 24) and healthy children (n = 16) by flow cytometry. Alterations in intracellular EG2-epitope expression were used to measure... [ABSTRACT FROM AUTHOR]

The proliferative and differentiative response of neutrophils to granulocyte–macrophage colony-stimulating factor (GM-CSF) is known to be impaired in patients with myelodysplastic syndromes (MDS). To investigate the mechanisms of the defective response in MDS, we examined expression levels of GM-CSF receptor α (GMRα) and common β (βc) subunits on CD16+ neutrophils, CD14+ monocytes and CD3+ T cells from 26 MDS patients and 10 healthy controls using flow cytometry. Expression of GMRα was significantly decreased on the neutrophils of five out of 26 patients and was not specific for any FAB subtype. In contrast, βc expression on neutrophils was significantly reduced in 14 out of 26 patients with a higher proportion occurring in the advanced stages of MDS including refractory anaemia with excess of blasts (RAEB), RAEB in transformation (RAEBt) and overt leukaemia compared with refractory anaemia (RA)/RA with ringed sideroblasts (RARS) or healthy controls. Decreased βc also correlated with the degree of hypogranular neutrophil morphology and increased infection. Expression of both subunits on T cells and monocytes in MDS was similar to normal controls. Polymerase chain reaction amplification of reverse-transcribed mRNA isolated from the affected neutrophils suggests that the reduction of βc may result from decreased message levels. The observed reduction in GM-CSF receptor expression could account for the impaired proliferative and maturational responses in MDS. [ABSTRACT FROM AUTHOR]

Langerhans' cell histiocytosis (LCH) is characterized by the proliferation of large mononucleated cells containing Birbeck granules and expressing CD1a. Recent studies have demonstrated that LCH is a clonal proliferation; however, its aetiology is still unknown. Growth and differentiation of bone-marrow-derived cells are controlled by cytokines. The proliferation, differentiation and activation of normal Langerhans cells are controlled by granulocyte/macrophage colony-stimulating factor (GM-CSF) in vitro. Therefore, GM-CSF could be implicated in the pathogenesis of LCH. Indeed, LCH cells contain GM-CSF, and children with disseminated LCH have an elevated GM-CSF serum level. As a cytokine only acts on cells expressing a specific receptor, we investigated the presence of GM-CSF receptor on LCH cells. Fourteen frozen tissue samples from children with LCH were studied by in situ immunohistochemistry with two mouse monoclonal antibodies specific for the alpha chain of the GM-CSF receptor (CDw116). LCH cells of all the samples were positively stained with both antibodies. This study suggests that GM-CSF may be a growth factor for LCH cells. [ABSTRACT FROM AUTHOR]

We investigated the interaction between GM-CSF and its receptor on human granulocytes and on several human tumor cell lines. Specific high-affinity binding for GM-CSF was characterized by Scatchard plot analysis. The specific radioactivity of the I-labeled derivative of rH. GM-CSF was determined by self-displacement analysis and calculated to be 30 μCi/μg. The maximum concentration of binding sites (B max) in granulocytes was 40 fmol/mg protein (2,200 molecules GM-CSF bound/cell) and the dissociation constant (K) was 0.42 nM. No binding sites for GM-CSF were found in two lung cancer cell lines, SCLC-16HV and NCI-N417 or in the urinary bladder carcinoma cell line 5637, whereas the promyelocytic leukemia cell line HL60 was positive for GM-CSF binding. Time course experiments showed maximum binding of GM-CSF in granulocytes after an incubation period of 60 min and a decrease in binding after an incubation period of 2 h. In parallel, we found a maximum biological signal when granulocytes were preincubated for 90 min with GM-CSF, and a decrease after an incubation time of 120 min. Preincubation of the cells with rH. GM-CSF induced an enhancement of the production of activated oxygen species by the cells in response to PMA. [ABSTRACT FROM AUTHOR]

A factor-dependent human hemopoietic cell line, TF-1, requires interleukln 3 (IL-3) or granulocyte/acrophage colony-stimulating factor (GM-CSF) for its long-term growth. We have found that IL-4, IL-5, and IL-6 also support the growth of TF-1 and that IL-1 enhances the proliferative effect of these cytoklnes. Augmentation by IL-1 is associated with up-regulatlon of the receptors for IL-3, IL-5, GM-CSF, and erythropoietln (Epo). IL-1 increased the number of binding sites forIL-3 and Epo without changing their affinities. In contrast, IL-1 Increased the number of high affinity binding sites forGM-CSF and IL-5, whereas the total number of binding sites was unchanged. Chemical crossllnking experiments Indicated that the receptors for IL-3, IL-5, and GM-CSF were composed of two components and that the molecular masses of the larger components of these cytokine receptors were quite similar (120 kd). The enhanced expression of the larger components of theIL-3, IL-5, and GM-CSF receptors by IL-1 may be responsible for IL-1-induced up-regulation of these receptors. These observations are consistent with the model that the receptors for IL-3 and GM-CSF share a common component. [ABSTRACT FROM PUBLISHER]

Abstract: In contrast to leukocytosis, paraneoplastic hypereosinophilia is uncommon in lung cancer. We present a patient with large-cell carcinoma of the lung, in which cancer cells generate large amounts of GM-CSF leading to a leukemoid reaction with prominent hypereosinophilia and potentially involved in autocrine tumor stimulation. [Copyright &y& Elsevier]