Granulocyte-macrophage Colony-stimulating Factor Inhibition Research Articles

Decreased expression of airway epithelial Axl is associated with eosinophilic inflammation in severe asthma

BackgroundAirway epithelium-derived cytokines are critical to provoke and perpetuate type 2 inflammation in asthma. Yet it is poorly understood how this epithelial cell-driven inflammatory response is negatively regulated. We previously reported that Axl receptor tyrosine kinase was expressed by basal cells in the airway epithelium and had a role in defining their stem cell identity. However, whether and how Axl regulates airway type 2 inflammation remains unknown. MethodsWe performed immunofluorescence staining to compare Axl expression in airway epithelium between non-asthmatic subjects, mild-moderate asthma and severe asthma. We confirmed this result by interrogating public databases of global gene expression in endobronchial biopsies. We then quantified eosinophil numbers infiltrating into the trachea of wild-type or Axl-knockout mice that were intranasally treated with house dust mite extracts (HDM). Cell-based assays using siRNA targeting Axl were further performed to identify molecules involved in Axl-mediated regulation of inflammation. ResultsHistological assessments and transcriptome analyses revealed decreases in protein and mRNA of Axl in airway basal cells of severe asthmatics. This reduction of Axl expression was correlated with infiltration of eosinophils and mast cells in severe asthmatics. Eosinophil infiltration was more evident in the trachea of Axl-knockout mice in response to repetitive HDM administration. siRNA-mediated knockdown of Axl increased mRNA and protein expression of granulocyte macrophage-colony stimulating factor (GM-CSF) in human bronchial epithelial cells. ConclusionsAxl kinase expressed by basal cells may suppress excessive eosinophilic inflammation via inhibition of GM-CSF in the airway. Axl reduction has clinical implications for the pathogenesis of severe asthma.

Abstract 14310: Anti-GM-CSF Monoclonal Antibody Gimsilumab Improved Ventilator-Free Survival, Decreased Hospitalization Length, and Prevented NT-proBNP Rise in Invasively Ventilated Patients With Hyperinflammatory COVID-19 Pneumonia: A Subgroup Analysis From the Breathe Trial Suggests Neurohormonal Role for GM-CSF Inhibition

Background: Granulocyte-macrophage colony-stimulating factor (GM-CSF), a myeloid cell growth factor and pro-inflammatory cytokine, may drive the overactive host immune response in COVID-19. We conducted a clinical trial assessing the anti-GM-CSF monoclonal antibody gimsilumab for hyperinflammatory COVID-19 pneumonia (BREATHE). Here, we report a pre-specified subgroup analysis demonstrating a signal of benefit in patients invasively ventilated at baseline. Methods: BREATHE (NCT04351243) was a double-blind, randomized, placebo-controlled trial at 21 US locations. Patients were randomized 1:1 to receive two doses of IV gimsilumab or placebo one week apart. The study included hospitalized COVID-19 patients with hyperinflammation (CRP ≥50 mg/L or ferritin ≥1,000 ng/mL) and pre-ARDS lung injury or ARDS. The primary endpoint was all-cause mortality at day 43, and key secondary outcomes assessed ventilator use and hospitalization length. Results: 225 patients were randomized and dosed. 41 patients were invasively ventilated at baseline. Steroid use and baseline characteristics were generally balanced across study arms in this subgroup. Ventilated patients treated with gimsilumab demonstrated improvements over placebo on the primary and key secondary endpoints (Table 1). Contrasting the placebo group, gimsilumab-treated patients did not experience a sharp rise in NT-proBNP, a marker of heart failure, through day 43 (Figure 1). Conclusions: GM-CSF inhibition may be therapeutic in ventilated COVID-19 patients through a neurohormonal mechanism. More studies are needed to assess the role of GM-CSF in COVID-19-associated cardiomyopathy, volume status, and ARDS.

GM-CSF-based treatments in COVID-19: reconciling opposing therapeutic approaches.

Therapeutics against coronavirus disease 2019 (COVID-19) are urgently needed. Granulocyte–macrophage colony-stimulating factor (GM-CSF), a myelopoietic growth factor and pro-inflammatory cytokine, plays a critical role in alveolar macrophage homeostasis, lung inflammation and immunological disease. Both administration and inhibition of GM-CSF are currently being therapeutically tested in COVID-19 clinical trials. This Perspective discusses the pleiotropic biology of GM-CSF and the scientific merits behind these contrasting approaches.

Effects of Granulocyte Macrophage Colony-Stimulating Factor Inhibition on the Skin/Nerve Cell Model In Vitro.

The present study is based on the concept of neuro-aging and how it may affect surrounding skin cells. It has been shown that many factors play a significant role in skin homeostasis by interfering with various cytokines, either through activation or inhibition. Granulocyte macrophage colony-stimulating factor (GM-CSF) is generally recognized as an inflammatory cytokine, and our previous study has shown its effects on neuronal senescence after ultraviolet (UV) irradiation of skin cells. Following our previous work, this study was performed to investigate the neuroprotective effects of a GM-CSF antagonist, and how it may play an essential role in mediating anti-senescence and anti-inflammatory effects in the keratinocyte/nerve aging model. When human blastoma cells (SH-SY5Y) were treated with 10 ng/ml of GM-CSF, the levels of regulatory RNAs associated with aging, such as matrix metalloproteinase-9 (MMP9), nuclear factor NF-kappa-B p50 subunit (NFKB), inducible nitric oxide synthase (iNOS), and interleukin 1 beta (IL-1β) increased, whereas GM-CSF inhibition caused their expression to decrease. A decrease in the antioxidant, glutathione (GSH) was observed after SH-SY5Y cells were treated with GM-CSF. This study confirms that this GM-CSF antagonist may play an important role in neural senescence, where inhibition may be a new target in the skin/nerve aging model.

GM-CSF inhibition reduces cytokine release syndrome and neuroinflammation but enhances CAR-T cell function in xenografts

AbstractChimeric antigen receptor T (CAR-T) cell therapy is a new pillar in cancer therapeutics; however, its application is limited by the associated toxicities. These include cytokine release syndrome (CRS) and neurotoxicity. Although the IL-6R antagonist tocilizumab is approved for treatment of CRS, there is no approved treatment of neurotoxicity associated with CD19-targeted CAR-T (CART19) cell therapy. Recent data suggest that monocytes and macrophages contribute to the development of CRS and neurotoxicity after CAR-T cell therapy. Therefore, we investigated neutralizing granulocyte-macrophage colony-stimulating factor (GM-CSF) as a potential strategy to manage CART19 cell–associated toxicities. In this study, we show that GM-CSF neutralization with lenzilumab does not inhibit CART19 cell function in vitro or in vivo. Moreover, CART19 cell proliferation was enhanced and durable control of leukemic disease was maintained better in patient-derived xenografts after GM-CSF neutralization with lenzilumab. In a patient acute lymphoblastic leukemia xenograft model of CRS and neuroinflammation (NI), GM-CSF neutralization resulted in a reduction of myeloid and T cell infiltration in the central nervous system and a significant reduction in NI and prevention of CRS. Finally, we generated GM-CSF–deficient CART19 cells through CRISPR/Cas9 disruption of GM-CSF during CAR-T cell manufacturing. These GM-CSFk/o CAR-T cells maintained normal functions and had enhanced antitumor activity in vivo, as well as improved overall survival, compared with CART19 cells. Together, these studies illuminate a novel approach to abrogate NI and CRS through GM-CSF neutralization, which may potentially enhance CAR-T cell function. Phase 2 studies with lenzilumab in combination with CART19 cell therapy are planned.

Granulocyte-Macrophage Colony-Stimulating Factor as a Therapeutic Target in Multiple Sclerosis

Multiple sclerosis is an inflammatory neurodegenerative disease of the central nervous system (CNS) and the most frequent cause of non-traumatic disability in adults in the Western world. Currently, several drugs have been approved for the treatment of multiple sclerosis. While the newer drugs are more effective, they have less favourable safety profiles. Thus, there is a need to identify new targets for effective and safe therapies, particularly in patients with progressive disease for whom no treatments are available. One such target is granulocyte-macrophage colony-stimulating factor (GM-CSF) or its receptor. In this article we review data on the potential role of GM-CSF and GM-CSF inhibition in MS. We discuss the expression and function of GM-CSF and its receptor in the CNS, as well as data from animal studies and clinical trials in MS.

Central inhibition of granulocyte-macrophage colony-stimulating factor is analgesic in experimental neuropathic pain.

With less than 50% of patients responding to the current standard of care and poor efficacy and selectivity of current treatments, neuropathic pain continues to be an area of considerable unmet medical need. Biological therapeutics such as monoclonal antibodies (mAbs) provide better intrinsic selectivity; however, delivery to the central nervous system (CNS) remains a challenge. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is well described in inflammation-induced pain, and early-phase clinical trials evaluating its antagonism have exemplified its importance as a peripheral pain target. Here, we investigate the role of this cytokine in a murine model of traumatic nerve injury and show that deletion of the GM-CSF receptor or treatment with an antagonizing mAb alleviates pain. We also demonstrate enhanced analgesic efficacy using an engineered construct that has greater capacity to penetrate the CNS. Despite observing GM-CSF receptor expression in microglia and astrocytes, the gliosis response in the dorsal horn was not altered in nerve injured knockout mice compared with wild-type littermate controls as evaluated by ionized calcium binding adapter molecule 1 (Iba1) and glial fibrillary acidic protein, respectively. Functional analysis of glial cells revealed that pretreatment with GM-CSF potentiated lipopolysaccharide-induced release of proinflammatory cytokines. In summary, our data indicate that GM-CSF is a proinflammatory cytokine that contributes to nociceptive signalling through driving spinal glial cell secretion of proinflammatory mediators. In addition, we report a successful approach to accessing CNS pain targets, providing promise for central compartment delivery of analgesics.

Abstract 3374: GM-CSF and MMP9 are key regulators of the effect of adipose progenitor cells over breast cancer onset and metastatic progression in obesity

Abstract We recently described a human cell population with progenitor-like phenotype (CD45-CD34+), resident in the white adipose tissue (WAT) and able to promote local and metastatic breast cancer (BC) progression and angiogenesis (Orecchioni et al., 2013). The molecular mechanism involved in this interaction has been so far elusive. An extensive screening of candidate molecules related to angiogenesis, inflammation, motility and invasiveness revealed that two proteins are significantly up-regulated in WAT-derived progenitors following culture with BC cells: Granulocyte-macrophage colony-stimulating factor (GM-CSF) and Matrix metallopeptidase 9 (MMP9). In vivo, both proteins were overexpressed in orthotopic models of human BC co-injected with human WAT progenitors. The inhibition of GM-CSF by a monoclonal antibody in diet-induced, obese BC mice led to a reduced intratumor vascularization and a strong impairment of WAT immunosuppressive microenvironment, targeting mainly myeloid cells such as macrophages and myeloid derived suppressor cells (MDSCs) in peritumoral WAT. Circulating levels of monocytes and of CD4+CD25brightCD127low/neg T-regulatory cells (T-regs) were also reduced in treated mice. Similarly, soluble immunosuppressive factors, such as IL-10 and IL-5, and CD274 (PD-L1) were reduced in tumors and WAT collected from immune competent mice neutralized for GM-CSF, confirming the crucial role of the factor in promoting tumor immune escape. This resulted in a significantly reduced local BC growth and lower metastatic progression in vivo. All these findings challenge the clinical use of GM-CSF. In the same syngeneic model, MMP9 inhibition reduced neoplastic angiogenesis and significantly decreased local and metastatic tumor growth, without altering immune cells composition in tumor microenvironment. The combined inhibition of GM-CSF and MMP9 was synergic in impairing angiogenesis, local and metastatic BC growth in diet-induced obese orthotopic BC models, indicating a potential complementary role in tumor spread. As we recently reported that Metformin targets both BC cells and the neoplastic WAT environment (Orecchioni et al., 2015), we investigated Metformin effect over GM-CSF and MMP9 expression. Metformin inhibited GM-CSF and MMP9 up-release from WAT progenitors in vitro. Circulating GM-CSF was significantly impaired in BC xenografts administered with Metformin and MMP9 expression was also affected by the treatment. Collectively these results indicate GM-CSF and MMP9 as key candidates involved in the pro-tumorigenic effect of WAT progenitors on BC in a setting of obesity. The comparison between Metformin and GM-CSF/MMP9 specific inhibition is currently under investigation. Citation Format: Francesca Reggiani, Valentina Labanca, Giovanna Talarico, Stefania Orecchioni, Patrizia Mancuso, Francesco Bertolini. GM-CSF and MMP9 are key regulators of the effect of adipose progenitor cells over breast cancer onset and metastatic progression in obesity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3374.

GM-CSF as a therapeutic target in autoimmune diseases.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been known as a hematopoietic growth factor and immune modulator. Recent studies revealed that GM-CSF also had pro-inflammatory functions and contributed to the pathogenicity of Th17 cells in the development of Th17-mediated autoimmune diseases. GM-CSF inhibition in some animal models of autoimmune diseases showed significant beneficial effects. Therefore, several agents targeting GM-CSF are being developed and are expected to be a useful strategy for the treatment of autoimmune diseases. Particularly, in clinical trials for rheumatoid arthritis (RA) patients, GM-CSF inhibition showed rapid and significant efficacy with no serious side effects. This article summarizes recent findings of GM-CSF and information of clinical trials targeting GM-CSF in autoimmune diseases.

Complementary action of granulocyte macrophage colony-stimulating factor and interleukin-17A induces interleukin-23, receptor activator of nuclear factor-κB ligand, and matrix metalloproteinases and drives bone and cartilage pathology in experimental arthritis: rationale for combination therapy in rheumatoid arthritis.

IntroductionType 17 T helper cells and interleukin (IL)-17 play important roles in the pathogenesis of human and murine arthritis. Although there is a clear link between IL-17 and granulocyte macrophage colony-stimulating factor (GM-CSF) in the inflammatory cascade, details about their interaction in arthritic synovial joints are unclear. In view of the introduction of GM-CSF and IL-17 inhibitors to the clinic, we studied how IL-17 and GM-CSF orchestrate the local production of inflammatory mediators during experimental arthritis.MethodsTo allow detection of additive, complementary or synergistic effects of IL-17 and GM-CSF, we used two opposing experimental approaches: treatment of arthritic mice with neutralising antibodies to IL-17 and GM-CSF and local overexpression of these cytokines in naive synovial joints. Mice were treated for 2 weeks with antibodies against IL-17 and/or GM-CSF after onset of collagen-induced arthritis. Naive mice were injected intraarticularly with adenoviral vectors for IL-17 and/or GM-CSF, resulting in local overexpression. Joint inflammation was monitored by macroscopic scoring, X-rays and histology. Joint washouts, synovial cell and lymph node cultures were analysed for cytokines, chemokines and inflammatory mediators by Luminex analysis, flow cytometry and quantitative polymerase chain reaction.ResultsCombined therapeutic anti-IL-17 and anti-GM-CSF ameliorated arthritis progression, and joint damage was dramatically reduced compared with treatment with anti-IL-17 or anti-GM-CSF alone. Anti-IL-17 specifically reduced synovial IL-23 transcription, whereas anti-GM-CSF reduced transcription of matrix metalloproteinases (MMPs) and receptor activator of nuclear factor κB ligand (RANKL). Overexpression of IL-17 or GM-CSF in naive knee joints elicited extensive inflammatory infiltrate, cartilage damage and bone destruction. Combined overexpression revealed additive and synergistic effects on the production of MMPs, RANKL and IL-23 in the synovium and led to complete destruction of the joint structure within 7 days.ConclusionsIL-17 and GM-CSF differentially mediate the inflammatory process in arthritic joints and show complementary and local additive effects. Combined blockade in arthritic mice reduced joint damage not only by direct inhibition of IL-17 and GM-CSF but also by indirect inhibition of IL-23 and RANKL. Our results provide a rationale for combination therapy in autoinflammatory conditions, especially for patients who do not fully respond to inhibition of the separate cytokines.

OP0112 Synergism between GM-CSF and IL-17 Causes Enhanced Joint Pathology via the Production of IL-6 and IL-23

BackgroundT helper-17 (Th17) cells are important mediators of inflammatory diseases, and are the main pathogenic cell type in many animal models of autoimmunity. Recent studies highlight a surprising role for...

1.56 Synergism between GM-CSF and IL-17 causes enhanced joint pathology via the production of IL-6 and IL-23

Background and ObjectiveT helper-17 (Th17) cells are important mediators of inflammatory diseases, and are the main pathogenic cell type in many animal models of autoimmunity. Recent studies highlight a surprising...

Lung microenvironment contributes to the resistance of alveolar macrophages to develop tolerance to endotoxin*

Endotoxin tolerance corresponds to reprogramming of mononuclear phagocytes after iterative encounters with toll-like receptor agonists aimed to dampen the inflammatory response. We investigated why this phenomenon cannot be observed with murine alveolar macrophages. Animal study. Research institution laboratory. rag2-/-, rag2γc-/-, cd3ε-/-, µ-/-, il-15-/-, Jα18-/-, ifnγr-/-, il-18r-/-, and wild-type mice. Alveolar macrophages were harvested from untreated mice or after injection of endotoxin. Alveolar macrophages were activated in vitro with endotoxin (lipopolysaccharide), and tumor necrosis factor production was monitored. In contrast to monocytes or peritoneal macrophages, alveolar macrophages did not display endotoxin tolerance in an ex vivo model after injection of endotoxin. An in vivo systemic inhibition of granulocyte-macrophage colony-stimulating factor or interferon-γ allowed the induction of alveolar macrophage endotoxin tolerance, which was also observed in interferon-γ receptor-deficient mice. Using mice missing different leukocyte subsets and adoptive cell transfers, we demonstrated the involvement of B lymphocytes in interferon-γ production within the lung microenvironment and in the prevention of alveolar macrophage endotoxin tolerance. Furthermore, we demonstrated the importance of interleukin-18 in preventing alveolar macrophage endotoxin tolerance through studies of interleukin-18 messenger RNA expression in il-18r-/- mice and injection of interleukin-18 in rag2-/- and µ-/- mice. Our results support the conclusion that at homeostasis in the lungs, constitutive expression of granulocyte-macrophage colony-stimulating factor, interleukin-18, interferon-γ and possibly interleukin-15, and a cross-talk between B lymphocytes and alveolar macrophages create a microenvironment specific to the lungs that prevents alveolar macrophages from becoming tolerant to endotoxin.

Toll-like receptor 2/6 stimulation promotes angiogenesis via GM-CSF as a potential strategy for immune defense and tissue regeneration

AbstractToll-like receptors (TLRs) are known primarily as pathogen recognition receptors of the innate immunity, initiating inflammatory pathways to organize the immune defense. More recently, an involvement of TLRs in various physiologic and pathologic processes has been reported. Because many of these processes implicate angiogenesis, we here elucidated the role of a TLR2/6-dependent pathway on angiogenesis using the TLR2/6 agonist macrophage-activating lipopeptide of 2 kDa (MALP-2), a common bacterial lipopeptide. In vivo and in vitro Matrigel assays demonstrated that MALP-2 promoted angiogenesis in a TLR2/6-dependent manner. Moreover, MALP-2 induced endothelial cell proliferation and migration and a strong secretion of granulocyte-macrophage colony-stimulating factor (GM-CSF). GM-CSF release in response to MALP-2 from isolated vascular segments was completely prevented when the endothelium was removed. MALP-2 containing Matrigel implants exhibited vascular structures as well as CD45+ cells. MALP-2 induced migration of leukocytes and likewise GM-CSF release, particularly from the monocyte population. Inhibition of GM-CSF by siRNA or antibodies suppressed MALP-2-induced angiogenesis in vitro and in vivo. These results clearly identified a TLR2/6-dependent induction of angiogenesis by the bacterial lipopeptide MALP-2, which is mediated by GM-CSF. This might represent a general mechanism to enhance or restore blood flow and recruit immune cells for pathogen defense and tissue regeneration.

Abstract 5593: Toll-like Receptor 2/6 Stimulation Promotes Angiogenesis via Induction of Endothelial GM-CSF Expression

Background: Toll-like receptors (TLRs) are known as pathogen recognition receptors of the innate immunity, initiating inflammatory pathways to organize the immune defense. More recently, an involvement of TLRs in various physiological and pathological processes has been reported. Since many of these processes implicate angiogenesis, we here elucidated the role of a TLR2/6-dependent pathway on angiogenesis using the specific TLR2/6 agonist MALP-2, a common bacterial lipopeptide. Methods and Results: MALP-2 induced angiogenesis in vivo (Matrigel implants, hemoglobin content, H&E staining, P<0.01, n=4–8) and tube formation in vitro (Matrigel, P<0.05, n=3–5) which could be inhibited with neutralizing antibodies against TLR2/6. Moreover, endothelial cells responded to MALP-2 with enhanced proliferation and migration (BrdU-incorporation, transwell, P<0.01, n=5–7). Protein array revealed a strong secretion of granulocyte-macrophage colony stimulating factor (GM-CSF) from endothelial cells which was confirmed by ELISA (P<0.01, n=4) and could be inhibited with antibodies against TLR2/6. By contrast, smooth muscle cells and fibroblasts did not secrete GM-CSF in response to MALP-2 (ELISA, n=3–6). In addition, GM-CSF release from isolated vascular segments following MALP-2 stimulation was completely prevented when the endothelium was removed mechanically (ELISA, P<0.01, n=4). Capillaries generated by MALP-2 treatment contained erythrocytes as well as CD45 + cells (Matrigel implants, CD45 immunostaining, n=4). Following MALP-2 stimulation CD45 + cells showed likewise enhanced migration (transwell, P<0.05, n=5) and GM-CSF release (ELISA, P<0.01, n=5) however to a lower extent compared to endothelial cells. Finally, inhibition of GM-CSF by siRNA in vitro (Matrigel, P<0.05, n=4) or by antibodies in vivo (Matrigel implants, P<0.01, n=4–8) inhibited the MALP-2 induced angiogenesis. Conclusion: These results demonstrate a TLR2/6-dependent induction of angiogenesis by the bacterial lipopeptide MALP-2 which is mediated by GM-CSF. This might represent a general mechanism to enhance or restore blood flow and recruit immune cells for pathogen defense and tissue regeneration.

Azithromycin and Clarithromycin Inhibit Lipopolysaccharide-Induced Murine Pulmonary Neutrophilia Mainly through Effects on Macrophage-Derived Granulocyte-Macrophage Colony-Stimulating Factor and Interleukin-1β

Macrolide antibiotics possess immunomodulatory/anti-inflammatory properties. These properties are considered fundamental for the efficacy of macrolide antibiotics in the treatment of chronic inflammatory diseases like diffuse panbronchiolitis and cystic fibrosis. However, the molecular mechanisms and cellular targets of anti-inflammatory/immunomodulatory macrolide activity are still not fully understood. To describe anti-inflammatory effects of macrolides in more detail and to identify potential biomarkers of their activity, we have investigated the influence of azithromycin and clarithromycin on the inflammatory cascade leading to neutrophil infiltration into lungs after intranasal lipopolysaccharide challenge in mice. Azithromycin and clarithromycin pretreatment reduced total cell and neutrophil numbers in bronchoalveolar lavage fluid and myeloperoxidase concentration in lung tissue. In addition, concentrations of several inflammatory mediators, including CCL2, granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-1beta (IL-1beta), tumor necrosis factor alpha, and sE-selectin in lung homogenates were decreased after macrolide treatment. Inhibition of cytokine production observed in vivo was also corroborated in vitro in lipopolysaccharide-stimulated monocytes/macrophages, but not in an epithelial cell line. In summary, results presented in this article confirm that macrolides can suppress neutrophil-dominated pulmonary inflammation and suggest that the effect is mediated through inhibition of GM-CSF and IL-1beta production by alveolar macrophages. Besides GM-CSF and IL-1beta, CCL2 and sE-selectin are also identified as potential biomarkers of macrolide anti-inflammatory activity in the lungs.

Expression of CD73/ecto-5'-nucleotidase on human gingival fibroblasts and contribution to the inhibition of interleukin-1alpha-induced granulocyte-macrophage colony stimulating factor production.

CD73/5'-nucleotidase (5'-NT) is an ectoenzyme that participates in immune/inflammatory reactions. We examined the possible expression of CD73/5'-NT on human gingival fibroblasts (hGF), which are important to the immune/inflammatory system in periodontal tissue. We demonstrated that CD73/5'-NT was expressed on hGF by flow cytometry. We found that pre-treatment of hGF with 5'-AMP induced marked inhibition of granulocyte-macrophage colony-stimulating factor (GM-CSF) production from hGF upon stimulation with interleukin-1alpha (IL-1alpha) by enzyme-linked immunosorbent assay (ELISA). A specific inhibitor of 5'-NT, adenosine 5'-[alpha,beta-methylene] diphosphate blocked the inhibition of GM-CSF production, suggesting that adenosine converted from 5'-AMP acts on the inhibitory effects. The GM-CSF inhibition suggested that A3 receptor might be involved. The rank order of agonists was found to be (N6-benzyl-5'-N-ethylcarboxamidoadenosine) A3 receptor agonist > or = (2-chloroadenosine) non-selective agonist > (CGS-21680) A2A receptor agonist > adenosine > or = (N6-cyclohexyladenosine) A1 agonist. Further support for the main role of A3 receptor was the binding A3 antagonist [9-chloro-2-(2-furanyl)-5-([phenylacetyl]amino)[1,2,4]-triazolo[1,5-c]quinazdine] reversed the effect of adenosine, but no significant reverse was observed by A1 (1,3-dipropyl-8-cyclopentylxanthine), A2 [3,7-dimethyl-1-(2-propargyl)xanthine], A2A[8-(3-chlorostyryl)caffeine], and A2B (alloxazine) antagonists. The CD73/5'-NT expression was increased upon stimulation with gamma-interferon, but not other stimulants such as tumor necrosis factor-alpha, IL-4, lipopolysaccharide from Porphyromonas gingivalis and Escherichia coli, and fimbriae from P. gingivalis, and this increase was correlated with the enhanced GM-CSF inhibition by 5'-AMP but not adenosine. These findings suggested that CD73/5'-NT on hGF exerts an anti-inflammatory effects in periodontal disease by conversion from 5'-AMP to adenosine.

Microglial cell cycle-associated proteins control microglial proliferation in vivo and in vitro and are regulated by GM-CSF and density-dependent inhibition.

Hyperplasia of microglia is one characteristic of reactive gliosis, which is observed in various pathologic conditions in the central nervous system (CNS). To investigate the regulation mechanisms of microglial proliferation, the effect of granulocyte macrophage colony-stimulating factor (GM-CSF) on the expression of cell cycle-associated proteins was examined in the microglial cell line GMI-M6-3. After GM-CSF administration, cyclins D1, E, and A and cyclin-dependent kinase inhibitor p21Cip1 were increased, and another cyclin-dependent kinase inhibitor, p27Kip1, was decreased with morphologic transformation into ameboid form. By contrast, downregulation of these cyclins and p21Cip1, and strong upregulation of p27Kip1 accompanied by ramification were observed with GM-CSF deprivation. We also found that GMI-M6-3 exhibited homotypic contact inhibition of proliferation without any morphologic transformation. The increase of p27Kip1 and the decrease of cyclin A were suggested to play an important role in microglial contact inhibition. In addition, the direct effect of p27Kip1 to inhibit microglial proliferation was demonstrated both in vitro and in vivo by overexpression of p27Kip1.

Granulocyte/Macrophage-Colony-stimulating Factor (GM-CSF) Regulates Lung Innate Immunity to Lipopolysaccharide through Akt/Erk Activation of NFκB and AP-1 in Vivo

The lung innate immune response to lipopolysaccharide (LPS) coordinates cellular inflammation, mediator, and protease release essential for host defense but deleterious in asthma, chronic obstructive pulmonary disease, and cystic fibrosis. In vitro, LPS signals to the transcription factors NFkappaB via TLR4, MyD88, and IL-1R-associated kinase (IRAK), to AP-1 by mitogen-activated protein (MAP) kinases, and via an alternate route in IRAK-deficient mice, but the in vivo lung signaling pathway(s) are not understood. We investigated the role of Akt and Erk1/2 as LPS intensely stimulates granulocyte/macrophage-colony-stimulating factor (GM-CSF) release, and neutralizing GM-CSF profoundly suppressed LPS-induced inflammation, suppressed expression and activity of lung proteases, significantly reduced GM-CSF and tumor necrosis factor alpha (TNFalpha) mRNA expression, and dampened nuclear localization of both NFkappaB (p50/65) and AP-1. LPS markedly activated Akt and Erk1/2, but not p38, in a GM-CSF-dependent manner in direct temporal association with NFkappaB and AP-1 activation. Pharmacological inhibition of Akt or Erk activation in LPS-treated tracheal explants ex vivo inhibited the release of GM-CSF. These data implicate GM-CSF-dependent activation of Akt in the amplification of this response and demonstrate the role of Erks rather than p38 in lung LPS inflammatory responses. Inhibition of GM-CSF may be of therapeutic benefit in inflammatory diseases in which LPS contributes to lung damage.

Transforming growth factor-beta(1) augments granulocyte-macrophage colony-stimulating factor-induced proliferation of umbilical cord blood CD34(+) cells with an associated tyrosine phosphorylation of STAT5.

Several investigators have reported that transforming growth factor (TGF)-beta(1) and granulocyte-macrophage colony-stimulating factor (GM-CSF) synergistically support cell proliferation. However, the mechanisms involved have not been elucidated. To clarify the mechanisms of the synergistic action of TGF-beta(1) and GM-CSF, we compared the activation states of STAT5 and mitogen-activated protein kinase in CD34(+) cells and in GM-CSF-dependent hematopoietic cell lines. Human CD34(+) cells and GM-CSF-dependent cell lines (FKH-1, YNH-1, and M-07e) were stimulated with 1.25 ng/mL GM-CSF and/or 0.25 ng/mL TGF-beta(1), and 1.25 ng/mL GM-CSF and/or 0.25 ng/mL, 0.025 ng/mL TGF-beta(1), respectively, and cell proliferation was analyzed by [3H]thymidine uptake. Expression of signal transduction proteins and their phosphorylation states were determined by Western blotting. TGF-beta(1) synergistically enhanced the GM-CSF-augmented growth of CD34(+) cells and FKH-1 cells, but inhibited the growth of YNH-1 and M-07e cells. Tyrosine phosphorylation of STAT5 induced by GM-CSF was enhanced by stimulation with the combination of TGF-beta(1) and GM-CSF (TGF-beta(1)/GM-CSF) compared with that induced by GM-CSF alone in CD34(+) cells and FKH-1 cells. However, combinations of TGF-beta(1)/GM-CSF caused inhibition of GM-CSF-induced tyrosine phosphorylation in M-07e cells. No significant difference was observed in mitogen-activated protein kinase activation between CD34(+) cells and FKH-1 cells stimulated with GM-CSF/TGF-beta(1) or GM-CSF alone. Results suggest that TGF-beta(1) may augment GM-CSF-induced proliferation of CD34(+) cells in association with enhanced tyrosine phosphorylation of STAT5. Our data suggest a novel mechanism for the synergistic enhancement of cellular growth induced by the combination of TGF-beta(1) and GM-CSF.