ABSTRACT
BP180 (also termed type XVII collagen) is a hemidesmosomal protein and plays a critical role in cell-cell matrix adhesion in the skin; however, its other biological functions are largely unclear. In this study, we generated a BP180 functional-deficient mouse strain by deleting its extracellular domain of humanized NC16A (termed ΔNC16A mice). We found that BP180 is expressed by bone marrow mesenchymal stem cells (BM-MSC), and its functional deficiency leads to myeloid hyperplasia. Altered granulopoiesis in ΔNC16A mice is through bone marrow stromal cells evidenced by bone marrow transplantation. Furthermore, the level of G-CSF in bone marrow and circulation were significantly increased in ΔNC16A mice as compared with wild-type mice. The increased G-CSF was accompanied by an increased activation of the NF-κB signaling pathway in bone marrow and BM-MSC of ΔNC16A mice. Blockade of G-CSF restored normal granulopoiesis in ΔNC16A mice. Inhibition of NF-κB signaling pathway significantly reduces the release of G-CSF from ΔNC16A BM-MSC in vitro and the level of serum G-CSF in ΔNC16A mice. To our knowledge, these findings provide the first direct evidence that BP180 plays an important role in granulopoiesis through regulating NF-κB signaling pathway in BM-MSC.
Subject(s)
Autoantigens/metabolism , Bone Marrow/pathology , Leukopoiesis/immunology , Mesenchymal Stem Cells/metabolism , Neutrophils/physiology , Non-Fibrillar Collagens/metabolism , Animals , Autoantigens/genetics , Bone Marrow/drug effects , Bone Marrow/metabolism , Cell Differentiation/immunology , Disease Models, Animal , Granulocyte Colony-Stimulating Factor/antagonists & inhibitors , Granulocyte Colony-Stimulating Factor/blood , Granulocyte Colony-Stimulating Factor/metabolism , Humans , Hyperplasia/genetics , Hyperplasia/immunology , Mice , Mice, Transgenic , NF-kappa B/metabolism , Non-Fibrillar Collagens/genetics , Protein Domains/genetics , Signal Transduction/drug effects , Signal Transduction/immunology , Collagen Type XVIIABSTRACT
Neutrophils, the most abundant circulating leukocytes in human, play an indispensable role in the innate immune response to microbial infections. However, the contribution of tumor-associated neutrophils (TANs) to cancer progression and tumor immunity has been a matter of debate for decades. A higher neutrophil-to-lymphocyte ratio is associated with adverse overall survival in many solid tumors. Preclinical evidence exists to support both anti-tumor and pro-tumor activities of TANs, and TANs employ diverse mechanisms to influence tumor progression and metastasis. Here, we focus our review on the immunosuppressive mechanism of TANs and highlight how neutrophils can operate to dampen both innate and adaptive immunity to promote tumorigenesis. Here we discuss the intriguing and sometimes controversial connection between TANs and granulocytic/polymorphonuclear myeloid-derived suppressor cells (G/PMN-MDSCs). The molecular mechanisms underlying neutrophils' role in immunosuppression provide potential therapeutic targets for cancer treatment, either as monotherapies or as a part of combinatorial regimens. Therefore, we also highlight a number of neutrophil-targeting approaches that may improve the efficacy of current anticancer therapies, especially cancer immunotherapy. Currently interest is surging in the understanding and targeting of immunosuppressive neutrophils, with the goal of developing novel therapeutic strategies in the battle against cancer.
Subject(s)
Immune Tolerance , Neoplasms/immunology , Neutrophils/physiology , Extracellular Traps/physiology , Granulocyte Colony-Stimulating Factor/antagonists & inhibitors , Humans , Neoplasms/drug therapy , Neutrophils/drug effects , Proto-Oncogene Proteins c-met/antagonists & inhibitors , Transforming Growth Factor beta/antagonists & inhibitors , Tumor Microenvironment/immunologyABSTRACT
Bone loss in Staphylococcus aureus (S. aureus) osteomyelitis poses a serious challenge to orthopedic treatment. The present study aimed to elucidate how S. aureus infection in bone might induce bone loss. The C57BL/6 mice were injected with S. aureus (106 CFU/ml, 100 µl) or with the same amount of vehicle (control) via the tail vein. Microcomputed tomography (microCT) analysis showed bone loss progressing from week 1 to week 5 after infection, accompanied by a decreased number of osteocalcin-positive stained osteoblasts and the suppressed mRNA expression of Runx2 and osteocalcin. Transcriptome profiles of GSE30119 were downloaded and analyzed to determine the differences in expression of inflammatory factors between patients with S. aureus infected osteomyelitis and healthy controls, the data showed significantly higher mRNA expression of granulocyte colony-stimulating factor (G-CSF) in the whole blood from patients with S. aureus infection. Enzyme-linked immunosorbent assay (ELISA) analysis confirmed an increased level of G-CSF in the bone marrow and serum from S. aureus infected mice, which might have been due to the increased amount of F4/80+ macrophages. Interestingly, G-CSF neutralizing antibody treatment significantly rescued the bone loss after S. aureus infection, as evidenced by its roles in improving BV/TV and preserving osteocalcin- and osterix-positive stained cells. Importantly, we found that G-CSF level was significantly up-regulated in the serum from osteomyelitis patients infected by S. aureus Together, S. aureus infection might suppress the function of osteoblastic cells and induce progressive bone loss by up-regulating the level G-CSF, suggesting a therapeutic potential for G-CSF neutralization in combating bone loss in S. aureus osteomyelitis.
Subject(s)
Bone Remodeling , Granulocyte Colony-Stimulating Factor/metabolism , Osteoblasts/metabolism , Osteomyelitis/metabolism , Staphylococcal Infections/metabolism , Tibia/metabolism , Adult , Animals , Antibodies, Neutralizing/pharmacology , Bone Remodeling/drug effects , Case-Control Studies , Disease Models, Animal , Female , Granulocyte Colony-Stimulating Factor/antagonists & inhibitors , Granulocyte Colony-Stimulating Factor/immunology , Humans , Male , Mice, Inbred C57BL , Osteoblasts/drug effects , Osteoblasts/microbiology , Osteomyelitis/diagnostic imaging , Osteomyelitis/drug therapy , Osteomyelitis/microbiology , Signal Transduction , Staphylococcal Infections/diagnostic imaging , Staphylococcal Infections/drug therapy , Staphylococcal Infections/microbiology , Tibia/diagnostic imaging , Tibia/drug effects , Tibia/microbiology , Time Factors , X-Ray MicrotomographyABSTRACT
PURPOSE OF REVIEW: The current review gives a concise and updated overview of the relative new field of anticytokine autoantibodies (ACAA) and associated infections with a focus on recent findings regarding clinical manifestions, diagnostic and treatments. RECENT FINDINGS: Several recent case reports of unusual presentations of patients with neutralizing autoantibodies to IFN-γ and granulocyt macrophage colony-stimulating factor and expand the spectrum of clinical manifestations and suggest that anticytokine-mediated acquired immunodeficiency causing susceptibility to infection may be underdiagnosed. There is an expanding geographical distribution of antigranulocyt macrophage colony-stimulating factor associated Cryptococcus gattii infection. The spectrum of identified infections in patients with neutralizing antibodies to IFN-γ has a strong endemic component. Rituximab or cyclophophamide in addition to antimycobacterials could be a treatment options in refractory cases. NF-κB2 deficiency may be associated with a complex pattern of high titre neutralizing ACAA similar to autoimmune polyglandular syndrome type I and Thymoma. New technique for the detection of anticytokine antibodies are presented. Quantiferon testing, which is widely available for TB-diagnostic, may be repurposed to detect anti-IFN-γ autoantibodies. We propose that this test could be as well used to show if they are neutralizing. SUMMARY: ACAA are an emerging cause of acquired immunodeficiency which is likely underdiagnosed. Recent case reports document expanding spectra of clinical manifestations. NF-κB2 deficiency may be associated with a complex anti cytokine autoantibody pattern.
Subject(s)
Autoantibodies/adverse effects , Cytokines/antagonists & inhibitors , Disease Susceptibility , Infections/diagnosis , Infections/etiology , Infections/therapy , Antibodies, Neutralizing/immunology , Autoantibodies/immunology , Cytokines/immunology , Disease Susceptibility/immunology , Genetic Predisposition to Disease , Granulocyte Colony-Stimulating Factor/antagonists & inhibitors , Granulocyte Colony-Stimulating Factor/immunology , Humans , Interferon-gamma/antagonists & inhibitors , Interferon-gamma/immunology , Mycobacterium Infections/diagnosis , Mycobacterium Infections/etiology , Mycobacterium Infections/therapy , NF-kappa B/deficiency , Opportunistic Infections/diagnosis , Opportunistic Infections/etiology , Opportunistic Infections/therapy , Severity of Illness IndexABSTRACT
Emergency hematopoiesis facilitates the rapid expansion of inflammatory immune cells in response to infections by pathogens, a process that must be carefully regulated to prevent potentially life threatening inflammatory responses. Here, we describe a novel regulatory role for the cytokine IFNγ that is critical for preventing fatal encephalitis after viral infection. HSV1 encephalitis (HSE) is triggered by the invasion of the brainstem by inflammatory monocytes and neutrophils. In mice lacking IFNγ (GKO), we observed unrestrained increases in G-CSF levels but not in GM-CSF or IL-17. This resulted in uncontrolled expansion and infiltration of apoptosis-resistant, degranulating neutrophils into the brainstem, causing fatal HSE in GKO but not WT mice. Excessive G-CSF in GKO mice also induced granulocyte derived suppressor cells, which inhibited T-cell proliferation and function, including production of the anti-inflammatory cytokine IL-10. Unexpectedly, we found that IFNγ suppressed G-CSF signaling by increasing SOCS3 expression in neutrophils, resulting in apoptosis. Depletion of G-CSF, but not GM-CSF, in GKO mice induced neutrophil apoptosis and reinstated IL-10 secretion by T cells, which restored their ability to limit innate inflammatory responses resulting in protection from HSE. Our studies reveals a novel, complex interplay among IFNγ, G-CSF and IL-10, which highlights the opposing roles of G-CSF and IFNγ in regulation of innate inflammatory responses in a murine viral encephalitis model and reveals G-CSF as a potential therapeutic target. Thus, the antagonistic G-CSF-IFNγ interactions emerge as a key regulatory node in control of CNS inflammatory responses to virus infection.
Subject(s)
Cell Proliferation/genetics , Central Nervous System/virology , Chemotaxis, Leukocyte/genetics , Encephalitis, Viral/prevention & control , Granulocyte Colony-Stimulating Factor/antagonists & inhibitors , Interferon-gamma/physiology , Neutrophils/physiology , Animals , Cell Proliferation/drug effects , Cells, Cultured , Central Nervous System/drug effects , Central Nervous System/metabolism , Chemotaxis, Leukocyte/drug effects , Encephalitis, Viral/genetics , Encephalitis, Viral/immunology , Female , Granulocyte Colony-Stimulating Factor/pharmacology , Granulocyte Colony-Stimulating Factor/physiology , Interferon-gamma/genetics , Interferon-gamma/pharmacology , Male , Mice , Mice, Knockout , Neutrophils/drug effectsABSTRACT
The survival rate for pancreatic ductal adenocarcinoma (PDAC) remains low. More therapeutic options to treat this disease are needed, for the current standard of care is ineffective. Using an animal model of aggressive PDAC (Kras/p48TGFßRIIKO), we discovered an effect of TGFß signaling in regulation of G-CSF secretion in pancreatic epithelium. Elevated concentrations of G-CSF in PDAC promoted differentiation of Ly6G+ cells from progenitors, stimulated IL10 secretion from myeloid cells, and decreased T-cell proliferation via upregulation of Arg, iNOS, VEGF, IL6, and IL1b from CD11b+ cells. Deletion of csf3 in PDAC cells or use of a G-CSF-blocking antibody decreased tumor growth. Anti-G-CSF treatment in combination with the DNA synthesis inhibitor gemcitabine reduced tumor size, increased the number of infiltrating T cells, and decreased the number of Ly6G+ cells more effectively than gemcitabine alone. Human analysis of human datasets from The Cancer Genome Atlas and tissue microarrays correlated with observations from our mouse model experiments, especially in patients with grade 1, stage II disease. We propose that in aggressive PDAC, elevated G-CSF contributes to tumor progression through promoting increases in infiltration of neutrophil-like cells with high immunosuppressive activity. Such a mechanism provides an avenue for a neoadjuvant therapeutic approach for this devastating disease. Cancer Immunol Res; 5(9); 718-29. ©2017 AACR.
Subject(s)
Adenocarcinoma/genetics , Carcinoma, Pancreatic Ductal/genetics , Granulocyte Colony-Stimulating Factor/genetics , Proto-Oncogene Proteins p21(ras)/genetics , Transforming Growth Factor beta/genetics , Adenocarcinoma/immunology , Adenocarcinoma/pathology , Animals , Carcinoma, Pancreatic Ductal/immunology , Carcinoma, Pancreatic Ductal/pathology , Cell Proliferation/genetics , Disease Models, Animal , Disease Progression , Gene Expression Regulation, Neoplastic , Granulocyte Colony-Stimulating Factor/antagonists & inhibitors , Granulocyte Colony-Stimulating Factor/immunology , Granulocyte Colony-Stimulating Factor/metabolism , Humans , Interferon-Stimulated Gene Factor 3, gamma Subunit/genetics , Mice , Mice, Knockout , Signal Transduction/genetics , T-Lymphocytes/immunologyABSTRACT
It has been shown that Granulocyte colony-stimulating factor (G-CSF) has a higher expression in malignant tumors, and anti-G-CSF therapy considerably decreases tumor growth, tumor vascularization and metastasis. Thus, blocking the signaling pathway of G-CSF could be beneficial in cancer therapy. This study is aimed at designing and producing a monoclonal nanobody that could act as an antagonist of G-CSF receptor. Nanobodies are the antigen binding fragments of camelid single-chain antibodies, also known as VHH. These fragments have exceptional properties which makes them ideal for tumor imaging and therapeutic applications. We have used our previously built nanobody phage libraries to isolate specific nanobodies to the G-CSF receptor. After a series of cross-reactivity and affinity experiments, two unique nanobodies were selected for functional analysis. Proliferation assay, real-time PCR and immunofluorescence assays were used to characterize these nanobodies. Finally, VHH26 nanobody that was able to specifically bind G-CSF receptor (G-CSF-R) on the surface of NFS60 cells and efficiently block G-CSF-R downstream signaling pathway in a dose-dependent manner was selected. This nanobody could be further developed into a valuable tool in tumor therapy and it forms a basis for additional studies in preclinical animal models.
Subject(s)
Granulocyte Colony-Stimulating Factor/antagonists & inhibitors , Receptors, Granulocyte Colony-Stimulating Factor/metabolism , Single-Domain Antibodies/metabolism , Animals , Cell Line, Tumor , Humans , Mice , Neovascularization, Pathologic/metabolism , Signal Transduction , Single-Chain Antibodies/metabolismABSTRACT
Glucocorticoids promote neutrophilic inflammation, the mechanisms of which are poorly characterized. Using a lipopolysaccharide (LPS)-induced acute murine lung injury model, we determined the role of granulocyte colony-stimulating factor (G-CSF) in mouse lung neutrophil numbers in the absence and presence of dexamethasone, a potent glucocorticoid. G-CSF was blocked using a neutralizing antibody. Airway neutrophil numbers, cytokine levels, and lung injury parameters were measured. Glucocorticoid treatment maintained LPS-induced airway G-CSF while suppressing TNF and IL-6. The addition of anti-G-CSF antibodies enabled dexamethasone to decrease airway G-CSF, neutrophils, and lung injury scores. In LPS-challenged murine lungs, structural cells and infiltrating leukocytes produced G-CSF. In vitro using BEAS 2B bronchial epithelial cells, A549 lung epithelial cells, human monocyte-derived macrophages, and human neutrophils, we found that dexamethasone and proinflammatory cytokines synergistically induced G-CSF. Blocking G-CSF production in BEAS 2B cells using shRNAs diminished the ability of BEAS 2B cells to protect neutrophils from undergoing spontaneous apoptosis. These data support that G-CSF plays a role in upregulation of airway neutrophil numbers by dexamethasone in the LPS-induced acute lung injury model.
Subject(s)
Dexamethasone/pharmacology , Granulocyte Colony-Stimulating Factor/antagonists & inhibitors , Lipopolysaccharides/toxicity , Lung Injury/drug therapy , Lung/drug effects , Neutrophils/drug effects , Animals , Cells, Cultured , Cytokines/metabolism , Glucocorticoids/pharmacology , Humans , Lung/metabolism , Lung/pathology , Lung Injury/chemically induced , Lung Injury/metabolism , Macrophages/drug effects , Macrophages/metabolism , Mice , Mice, Inbred BALB C , Neutrophils/metabolism , Neutrophils/pathologyABSTRACT
Acute exposure to cadmium (Cd), a toxic heavy metal, causes systemic inflammation characterized by neutrophilia. To elucidate the mechanism of neutrophilia induced by Cd, we investigated the induction of granulocyte colony-stimulating factor (G-CSF), which regulates neutrophil production, in mice with acute Cd toxicity, and compared it with mice injected with lipopolysaccharide (LPS) as an inducer of general inflammatory responses. We injected BALB/c mice with Cd at 2.5 mg/kg i.p. or LPS at 0.5 mg/kg i.p. and sampled the peripheral blood and organs at time points up to 24 h. In Cd-treated mice, the peripheral neutrophil count increased steadily up to 24 h, whereas LPS-treated mice showed a more rapid increase with a peak at 12 h. The serum G-CSF level increased gradually to reach a plateau at 12-18 h in Cd-treated mice, but LPS-treated mice showed a marked increase, reaching a peak at 2-3 h. A gradual elevation of G-CSF mRNA expression up to 24 h was detected by real-time PCR in the livers of Cd-treated mice, but in LPS-treated mice its highest expression was observed in the liver with a rapid increase at 2 h. By in situ hybridization using G-CSF RNA probes, hepatic Kupffer cells were identified as G-CSF-producing cells in the liver. These results indicated that Cd has a characteristic effect of delayed induction of G-CSF in the liver, causing systemic inflammation accompanied by prolonged neutrophilia.
Subject(s)
Cadmium Poisoning/physiopathology , Chemical and Drug Induced Liver Injury/physiopathology , Granulocyte Colony-Stimulating Factor/antagonists & inhibitors , Leukocytosis/etiology , Liver/drug effects , Neutrophils/drug effects , Systemic Inflammatory Response Syndrome/etiology , Acute Disease , Animals , Cadmium/blood , Cadmium/metabolism , Cadmium/toxicity , Cadmium Poisoning/immunology , Cadmium Poisoning/metabolism , Cadmium Poisoning/pathology , Chemical and Drug Induced Liver Injury/immunology , Chemical and Drug Induced Liver Injury/metabolism , Chemical and Drug Induced Liver Injury/pathology , Endotoxins/toxicity , Environmental Pollutants/blood , Environmental Pollutants/metabolism , Environmental Pollutants/toxicity , Female , Gene Expression Regulation/drug effects , Granulocyte Colony-Stimulating Factor/blood , Granulocyte Colony-Stimulating Factor/genetics , Granulocyte Colony-Stimulating Factor/metabolism , Kupffer Cells/drug effects , Kupffer Cells/immunology , Kupffer Cells/metabolism , Kupffer Cells/pathology , Lipopolysaccharides/toxicity , Liver/metabolism , Liver/pathology , Liver/physiopathology , Mice, Inbred BALB C , Neutrophils/immunology , Systemic Inflammatory Response Syndrome/chemically induced , Systemic Inflammatory Response Syndrome/physiopathology , Time Factors , Tissue Distribution , ToxicokineticsABSTRACT
BACKGROUND: Photodynamic therapy (PDT) affects the immune system and tumor cells' secretory activity. Hypoxia may limit PDT effects. The aim of this study was to determine the influence of PDT with aminolevulinic acid (ALA-PDT) in a hypoxic-like microenvironment on the secretion of growth factors: GM-CSF (granulocyte and macrophage colony stimulating factor), G-CSF (granulocyte colony-stimulating factor) and FGF (fibroblast growth factor) by experimental models of colon cancer cells in vitro. METHODS: Sublethal doses of ALA-PDT (ALA of 1000 µM, light fluence 10J/cm(2), power density rate of 1.5mW/cm(2), wavelength 600-720nm) were administered to two colorectal cancer cell lines varying in malignancy potential: SW480 (local malice) and SW620 (high metastatic activity) under normoxia and hypoxia-like environment. Hypoxia-mimic conditions were achieved by adding environment cobalt (II) chloride. Concentrations of growth factors were analyzed with Bio-Plex Pro™ Assay. RESULTS: ALA-PDT amplified the secretion of GM-CSF by both cell lines. The decrease in secretion of G-CSF and FGF was noticed in the SW620. SW620 line cells secreted higher levels of FGF and G-CSF, while SW480 cells more actively released GM-CSF. Compared to normoxic condition, no differences in the secretion of these factors in a hypoxic-like environment were found. CONCLUSIONS: ALA-PDT increased GM-CSF secretion, which stimulates antitumor defense and decreased secretion of FGF and G-CSF-factors responsible for tumor progression. No differences in the effects of ALA-PDT in the hypoxic-like environment suggests that the beneficial results of PDT are also continued in the later stages of the reaction.
Subject(s)
Aminolevulinic Acid/pharmacology , Colonic Neoplasms/metabolism , Intercellular Signaling Peptides and Proteins/metabolism , Photochemotherapy/methods , Photosensitizing Agents/pharmacology , Anaerobiosis , Apoptosis , Cell Line, Tumor , Cell Survival , Cobalt/pharmacology , Dose-Response Relationship, Drug , Fibroblast Growth Factors/antagonists & inhibitors , Granulocyte Colony-Stimulating Factor/antagonists & inhibitors , Granulocyte-Macrophage Colony-Stimulating Factor/biosynthesis , Humans , HypoxiaABSTRACT
Granulocyte colony-stimulating factor (G-CSF) is a cytokine that is highly expressed in human and mouse colorectal cancers (CRC). We previously reported that G-CSF stimulated human CRC cell growth and migration, therefore in this study we sought to examine the therapeutic potential of anti-G-CSF treatment for CRC. G-CSF is known to mobilize neutrophils, however its impact on other immune cells has not been well examined. Here, we investigated the effects of therapeutic anti-G-CSF treatment on CRC growth and anti-tumor immune responses. C57BL/6 mice treated with azoxymethane/dextran sodium sulfate (AOM/DSS) to induce neoplasms were administered anti-G-CSF or isotype control antibodies three times a week for three weeks. Animals treated with anti-G-CSF antibodies had a marked decrease in neoplasm number and size compared to the isotype control group. Colon neutrophil and macrophage frequency were unchanged, but the number of macrophages producing IL-10 were decreased while IL-12 producing macrophages were increased. NK cells were substantially increased in colons of anti-G-CSF treated mice, along with IFNγ producing CD4(+) and CD8(+) T cells. These studies are the first to indicate a crucial role for G-CSF inhibition in promoting protective anti-tumor immunity, and suggest that anti-G-CSF treatment is a potential therapeutic approach for CRC.
Subject(s)
Antibodies, Neutralizing/pharmacology , Colonic Neoplasms/immunology , Colonic Neoplasms/therapy , Granulocyte Colony-Stimulating Factor/antagonists & inhibitors , Killer Cells, Natural/immunology , Macrophages/immunology , T-Lymphocytes/immunology , Animals , Antibodies, Neutralizing/immunology , Colonic Neoplasms/genetics , Colonic Neoplasms/metabolism , Female , Granulocyte Colony-Stimulating Factor/pharmacology , Mice , Mice, Inbred C57BLABSTRACT
Cytokines generated from macrophages contribute to pathogenesis of inflammation-associated diseases. Here we show that γ-tocotrienol (γ-TE), a natural vitamin E form, inhibits lipopolysaccharide (LPS)-induced interleukin (IL)-6 production without affecting tumor necrosis factor α (TNF-α), IL-10 or cyclooxygenase-2 (COX-2) up-regulation in murine RAW264.7 macrophages. Mechanistic studies indicate that nuclear factor κB (NF-κB), but not c-Jun NH(2)-terminal protein kinase, p38 or extracellular signal-regulated kinase mitogen-activated protein kinases (MAPKs), is important to IL-6 production and that γ-TE treatment blocks NF-κB activation. In contrast, COX-2 appears to be regulated by p38 MAPK in RAW cells, but γ-TE has no effect on LPS-stimulated p38 phosphorylation. Despite necessary for IL-6, NF-κB activation by TNF-α or other cytokines is not sufficient for IL-6 induction with exception of LPS. CCAAT/enhancer-binding protein (C/EBP) ß appears to be involved in IL-6 formation because LPS induces C/EBPß up-regulation, which parallels IL-6 production, and knockdown of C/EBPß with small interfering RNA results in diminished IL-6. LPS but not individual cytokines is capable of stimulating C/EBPß and IL-6 in macrophages. Consistent with its dampening effect on IL-6, γ-TE blunts LPS-induced up-regulation of C/EBPß without affecting C/EBPδ. γ-TE also decreases LPS-stimulated granulocyte colony-stimulating factor (G-CSF), a C/EBPß target gene. Compared with RAW264.7 cells, γ-TE shows similar or stronger inhibitory effects on LPS-triggered activation of NF-κB, C/EPBß and C/EBPδ and more potently suppresses IL-6 and G-CSF in bone marrow-derived macrophages. Our study demonstrates that γ-TE has antiinflammatory activities by inhibition of NF-κB and C/EBPs activation in macrophages.
Subject(s)
Anti-Inflammatory Agents/pharmacology , CCAAT-Enhancer-Binding Proteins/antagonists & inhibitors , Chromans/pharmacology , Granulocyte Colony-Stimulating Factor/antagonists & inhibitors , Interleukin-6/metabolism , Lipopolysaccharides/pharmacology , Macrophages/drug effects , NF-kappa B/antagonists & inhibitors , Vitamin E/analogs & derivatives , Animals , CCAAT-Enhancer-Binding Proteins/metabolism , Cells, Cultured , Cyclooxygenase 2/metabolism , Granulocyte Colony-Stimulating Factor/metabolism , Macrophages/metabolism , Mice , Mice, Inbred BALB C , NF-kappa B/metabolism , Signal Transduction , Vitamin E/pharmacologyABSTRACT
MEL1 (MDS1/EVI1-like gene 1/PRDM16), which was identified as a gene near the chromosomal breakpoint in t(1;3)(p36;q21)-positive human acute myeloid leukemia cells, belongs to the PRDI-BF1-RIZ1 homologous (PR) domain (PRDM) family of transcription repressors. The short form of MEL1 (MEL1S), which lacks the PR-domain at the N-terminus, is the main form expressed in t(1;3)(p36;q21)-positive acute myeloid leukemia cells. The overexpression of MEL1S blocks granulocyte colony-stimulating factor (G-CSF)-induced myeloid differentiation in interleukin-3-dependent murine myeloid L-G3 cells. In this study, we show that treatment with the histone deacetylase inhibitor trichostatin A abolished the blockade of myeloid differentiation in L-G3 cells overexpressing MEL1S. The expression of MEL1S containing mutated CtBP-interacting motif (CIM) in L-G3 cells still blocked the myeloid differentiation induced by G-CSF. We found that the small ubiquitin-related modifier (SUMO) motif (SM) at lysine 568 (VKAE) adjacent to the CIM was necessary to obtain the maximum transcriptional repressor activity of MEL1S. L-G3 cells expressing MEL1S, and bearing mutated CIM and SM differentiated into granulocytes in response to G-CSF; this indicated that both the SUMO modification at lysine 568 and CtBP binding were required for MEL1S-mediated transcriptional repression and blockade of differentiation, which might be relevant for the process of leukemogenesis.
Subject(s)
Bone Marrow/drug effects , Cell Differentiation/physiology , DNA-Binding Proteins/metabolism , Granulocyte Colony-Stimulating Factor/antagonists & inhibitors , Lysine/metabolism , Transcription Factors/metabolism , Cell Line , Histone Deacetylase Inhibitors/pharmacology , Humans , Hydroxamic Acids/pharmacology , SumoylationABSTRACT
This article reports an inhibitory effect of rapamycin on the lipopolysaccharide (LPS)-induced expression of both inducible nitric oxide synthase (iNOS) and granulocyte-colony stimulating factor (G-CSF) in macrophages and its underlying mechanism. The study arose from an observation that rapamycin inhibited the LPS-induced increase in octamer-binding factor-2 (Oct-2) protein levels through a mammalian target of rapamycin (mTOR)-dependent pathway in mouse RAW264.7 macrophages. As both iNOS and G-CSF are potential Oct-2 target genes, we tested the effect of rapamycin on their expression and found that it reduced the LPS-induced increase in iNOS and G-CSF mRNA levels and iNOS and G-CSF protein levels. Blocking of mTOR-signaling using a dominant-negative mTOR expression plasmid resulted in inhibition of the LPS-induced increase in iNOS and G-CSF protein levels, supporting the essential role of mTOR. Forced expression of Oct-2 using the pCG-Oct-2 plasmid overcame the inhibitory effect of rapamycin on the LPS-induced increase in iNOS and G-CSF mRNA levels. Chromatin immunoprecipitation assays showed that LPS enhanced the binding of Oct-2 to the iNOS and G-CSF promoters and that this effect was inhibited by pretreatment with rapamycin. Moreover, RNA interference knockdown of Oct-2 reduced iNOS and G-CSF expression in LPS-treated cells. The inhibitory effect of rapamycin on the LPS-induced increase in Oct-2 protein levels and on the iNOS and G-CSF mRNA levels was also detected in human THP-1 monocyte-derived macrophages. This study demonstrates that rapamycin reduces iNOS and G-CSF expression at the transcription level in LPS-treated macrophages by inhibiting Oct-2 expression.
Subject(s)
Gene Expression Regulation, Enzymologic/drug effects , Granulocyte Colony-Stimulating Factor/metabolism , Lipopolysaccharides , Macrophages/drug effects , Nitric Oxide Synthase Type II/metabolism , Octamer Transcription Factor-2/metabolism , Sirolimus/pharmacology , Animals , Cell Line , Down-Regulation , Granulocyte Colony-Stimulating Factor/antagonists & inhibitors , Granulocyte Colony-Stimulating Factor/genetics , Humans , Mice , Nitric Oxide Synthase Type II/antagonists & inhibitors , Nitric Oxide Synthase Type II/genetics , Promoter Regions, GeneticABSTRACT
The cross talk between host and pathogen starts with recognition of bacterial signatures through pattern recognition receptors (PRRs), which mobilize downstream signaling cascades. We investigated the role of the cytosolic adaptor caspase recruitment domain family, member 9 (CARD9) in tuberculosis. This adaptor was critical for full activation of innate immunity by converging signals downstream of multiple PRRs. Card9(-/-) mice succumbed early after aerosol infection, with higher mycobacterial burden, pyogranulomatous pneumonia, accelerated granulocyte recruitment, and higher abundance of proinflammatory cytokines and granulocyte colony-stimulating factor (G-CSF) in serum and lung. Neutralization of G-CSF and neutrophil depletion significantly prolonged survival, indicating that an exacerbated systemic inflammatory disease triggered lethality of Card9(-/-) mice. CARD9 deficiency had no apparent effect on T cell responses, but a marked impact on the hematopoietic compartment. Card9(-/-) granulocytes failed to produce IL-10 after Mycobacterium tuberculosis infection, suggesting that an absent antiinflammatory feedback loop accounted for granulocyte-dominated pathology, uncontrolled bacterial replication, and, ultimately, death of infected Card9(-/-) mice. Our data provide evidence that deregulated innate responses trigger excessive lung inflammation and demonstrate a pivotal role of CARD9 signaling in autonomous innate host defense against tuberculosis.
Subject(s)
Adaptor Proteins, Signal Transducing/immunology , Immunity, Innate/immunology , Tuberculosis/immunology , Adaptor Proteins, Signal Transducing/genetics , Animals , Antigen-Presenting Cells/drug effects , Antigen-Presenting Cells/immunology , Antigen-Presenting Cells/metabolism , Antigen-Presenting Cells/microbiology , Antitubercular Agents/therapeutic use , Apoptosis/genetics , CARD Signaling Adaptor Proteins , Chemokines/blood , Chemokines/metabolism , Cytokines/blood , Cytokines/metabolism , Female , Gene Expression/genetics , Genetic Predisposition to Disease/genetics , Granulocyte Colony-Stimulating Factor/antagonists & inhibitors , Hypersensitivity, Delayed/genetics , Hypersensitivity, Delayed/immunology , Immunity, Innate/genetics , Interleukin-10/metabolism , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Intracellular Signaling Peptides and Proteins/metabolism , Lectins, C-Type , Leukocyte Reduction Procedures , Lung/drug effects , Lung/immunology , Lung/metabolism , Lung/microbiology , Lung/pathology , Macrophage Activation/drug effects , Macrophage Activation/genetics , Macrophage Activation/immunology , Male , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Nerve Tissue Proteins/antagonists & inhibitors , Nerve Tissue Proteins/metabolism , Neutrophils/drug effects , Neutrophils/immunology , Neutrophils/metabolism , Neutrophils/pathology , Pneumonia/pathology , Pneumonia/therapy , Polysaccharides, Bacterial/pharmacology , Protein-Tyrosine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/metabolismSubject(s)
Arsenicals/therapeutic use , Hematopoietic Stem Cell Mobilization/methods , Leukemia, Promyelocytic, Acute/therapy , Oxides/therapeutic use , Peripheral Blood Stem Cell Transplantation/methods , Adult , Arsenic Trioxide , Combined Modality Therapy , Female , Granulocyte Colony-Stimulating Factor/antagonists & inhibitors , Humans , Leukemia, Promyelocytic, Acute/genetics , Leukemia, Promyelocytic, Acute/prevention & control , Nuclear Proteins/genetics , Promyelocytic Leukemia Protein , Receptors, CXCR4/antagonists & inhibitors , Receptors, Retinoic Acid/genetics , Retinoic Acid Receptor alpha , Secondary Prevention , Transcription Factors/genetics , Tumor Suppressor Proteins/geneticsABSTRACT
Granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) are well-recognized regulators of hematopoiesis and have an established role as growth factors in clinical practice. G-CSF and GM-CSF regulate myeloid cell production, differentiation and activation, and might also be important for driving inflammatory responses. Inappropriate engagement of this pathway could be a critical amplification mechanism when maladaptive immune responses predispose to autoimmunity and sterile tissue inflammation. We postulate that antagonism of G-CSF or GM-CSF could represent a novel therapeutic approach for a variety of autoimmune-mediated inflammatory diseases, including rheumatoid arthritis.
Subject(s)
Antirheumatic Agents/therapeutic use , Arthritis, Rheumatoid/drug therapy , Granulocyte Colony-Stimulating Factor/physiology , Granulocyte-Macrophage Colony-Stimulating Factor/physiology , Animals , Arthritis, Rheumatoid/immunology , Arthritis, Rheumatoid/metabolism , Disease Models, Animal , Drug Design , Granulocyte Colony-Stimulating Factor/antagonists & inhibitors , Granulocyte-Macrophage Colony-Stimulating Factor/antagonists & inhibitors , Humans , Inflammation/drug therapy , Inflammation/physiopathology , Inflammation Mediators/therapeutic use , Neutrophils/physiologyABSTRACT
Lenalidomide is an agent that has shown great activity in patients with multiple myeloma (MM). However, studies have suggested that this drug negatively affects subsequent stem cell collection. To investigate whether lenalidomide impairs stem cell mobilization and collection, we reviewed data for patients with MM who underwent mobilization with filgrastim. Predictors of mobilization failure were evaluated using logistic regression analysis. In 26 (9%) of 302 myeloma patients, stem cell mobilization failed. Mobilization failed in 25% of patients who had previously received lenalidomide, compared with 4% of patients who had not received lenalidomide (P < .001). In a multivariate analysis, prior lenalidomide use (odds ratio: 5.9; 95% confidence interval [CI]: 2.4-14.3) and mobilization more than 1 year after diagnosis (odds ratio: 4.6; 95% CI: 1.9-11.1) were significantly associated with failed mobilization. Twenty-one of 26 patients in whom mobilization with filgrastim failed underwent remobilization with chemotherapy and filgrastim; in 18 (86%) of these 21 patients, stem cells were successfully mobilized and collected. In patients with multiple myeloma, prior lenalidomide therapy is associated with failure of stem cell mobilization with filgrastim. Remobilization with chemotherapy and filgrastim is usually successful in these patients.
Subject(s)
Antineoplastic Agents/adverse effects , Bone Marrow/drug effects , Granulocyte Colony-Stimulating Factor/antagonists & inhibitors , Hematopoietic Stem Cell Mobilization/methods , Multiple Myeloma/blood , Peripheral Blood Stem Cell Transplantation , Thalidomide/analogs & derivatives , Adult , Aged , Angiogenesis Inhibitors/administration & dosage , Angiogenesis Inhibitors/adverse effects , Angiogenesis Inhibitors/therapeutic use , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Antineoplastic Combined Chemotherapy Protocols/administration & dosage , Benzylamines , Combined Modality Therapy , Cyclams , Cyclophosphamide/administration & dosage , Cyclophosphamide/pharmacology , Dexamethasone/administration & dosage , Doxorubicin/administration & dosage , Etoposide/administration & dosage , Etoposide/pharmacology , Female , Filgrastim , Granulocyte Colony-Stimulating Factor/administration & dosage , Granulocyte Colony-Stimulating Factor/pharmacology , Heterocyclic Compounds/administration & dosage , Heterocyclic Compounds/pharmacology , Humans , Ifosfamide/administration & dosage , Ifosfamide/pharmacology , Lenalidomide , Leukapheresis , Male , Middle Aged , Multiple Myeloma/drug therapy , Multiple Myeloma/radiotherapy , Multiple Myeloma/surgery , Recombinant Proteins , Retrospective Studies , Risk Factors , Thalidomide/administration & dosage , Thalidomide/adverse effects , Thalidomide/therapeutic use , Transplantation, Autologous , Treatment Failure , Vincristine/administration & dosageABSTRACT
AIMS: Ethyl pyruvate (EtP) may prolong survival and ameliorate organ dysfunction in a variety of models of critical illness, e.g. severe sepsis and acute respiratory syndrome, by modulation of the expression of inflammatory mediators. Here, we studied the effects of EtP on the reactions in and between human neutrophils and lung epithelial (A549) cells in vitro. MAIN METHODS: Neutrophil adhesion to, surface expression of ICAM-1 and VCAM-1 on, and release of IL-8 and G-CSF from A549 cells were measured by ELISA after stimulation with IL-1 beta or TNFalpha. KEY FINDINGS: After treatment of A549 cells with EtP, a substantial reduction in the cytokine-induced adhesion of neutrophils to monolayers was noted, whereas sodium pyruvate (NaP) conferred no reduction. Likewise, treatment with 2.5-10 mM EtP (but not NaP) reduced ICAM-1 and VCAM-1 expression in a dose-dependent fashion. The generation of cytokines of significance for adhesive and proliferative events in host defense, IL-8 and G-CSF, was also potently impaired by EtP. SIGNIFICANCE: Exposure of lung epithelial cells to 2.5-10 mM EtP inhibited the generation of inflammatory-regulating cytokines IL-8 and G-CSF, reduced ICAM-1 and VCAM-1 expression and impeded the adhesiveness of neutrophils to lung epithelial cells. These are reactions of significance for early inflammatory responses in the lung, suggesting a role for EtP as a treatment for acute pulmonary conditions.
Subject(s)
Cell Adhesion/physiology , Epithelial Cells/metabolism , Lung/metabolism , Pyruvates/pharmacology , Respiratory Mucosa/cytology , Respiratory Mucosa/metabolism , Cell Adhesion/drug effects , Cell Line, Tumor , Epithelial Cells/drug effects , Granulocyte Colony-Stimulating Factor/antagonists & inhibitors , Granulocyte Colony-Stimulating Factor/metabolism , Humans , Intercellular Adhesion Molecule-1/biosynthesis , Interleukin-1beta/antagonists & inhibitors , Interleukin-1beta/physiology , Interleukin-8/antagonists & inhibitors , Interleukin-8/metabolism , Lung/cytology , Lung/drug effects , Neutrophils/drug effects , Neutrophils/metabolism , Respiratory Mucosa/drug effects , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Tumor Necrosis Factor-alpha/physiology , Vascular Cell Adhesion Molecule-1/biosynthesisABSTRACT
One of the most promising new strategies for the development of efficacious cancer therapies relies on the targeted delivery of biopharmaceutical to the tumor environment by the use of selective and specific antibodies. The identification of accessible perivascular proteins selectively overexpressed in cancer tissue may facilitate the development of antibody-based biopharmaceutical administration. This approach is potentially highly selective and specific, combining the presence of tumor biomarkers readily accessible from the blood vessels and the high rate of angiogenesis characteristic of cancer tissues. We performed ex vivo perfusions of surgically resected human colon cancer using a reactive ester derivative of biotin, thus achieving a selective covalent modification of accessible proteins in vascular structures and stroma. After extraction and purification, biotinylated proteins were digested and the resulting peptides submitted to a comparative mass spectrometry-based proteomic analysis, revealing quantitative differences between normal and cancer colon. Sixty-seven of the total 367 proteins identified were found to be preferentially expressed at the tumor site. We generated human monoclonal antibodies against 2 potential tumor targets, NGAL and GW112, and we proved their selective expression in cancer colon and not or barely in healthy tissues. This article presents the first proteomic analysis of human colorectal cancer structures readily accessible from the tumor vasculature, revealing the overexpression of novel tumor antigens which may serve as selective targets for antibody-based imaging and therapeutic biomolecular strategies.