Primary Acute Myeloid Leukemic Blasts Display Constitutive Serine 585 Phosphorylation within the 14-3-3 Binding Motif of the GM-CSF/IL-3 Receptor Required for Survival Signalling.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 70-70 ◽  
Author(s):  
Daniel Thomas ◽  
Jason Powell ◽  
Szu-Hee Lee ◽  
Angel F. Lopez ◽  
Guthridge Mark
Keyword(s):  
Cell Survival ◽  
Adaptor Protein ◽  
Human Monocyte ◽  
Buffy Coat ◽  
Binding Motif ◽  
Beta Chain ◽  
Blood Monocytes ◽  
Gm Csf ◽  
Threonine Kinases ◽  
Acute Myeloid

Abstract Acute myeloid leukemia (AML) is characterised by defects in the regulation of hematopoietic cell survival, differentiation and proliferation. IL-3 and GM-CSF exert potent survival and proliferative effects on a range of myeloid progenitor cells. Previously we have identified a novel cytoplasmic motif of the human GM-CSF/IL-3 receptor beta chain responsible for coordinating survival and proliferative responses in primary hematopoietic cells. The motif contains a serine residue (585) that is phosphorylated in response to cytokine and couples to the PI3K/AKT pathway via the 14-3-3 adaptor protein and is required for cell survival (Guthridge et al, Molecular Cell6:99, 2000;Blood103:820, 2004). An adjacent tyrosine residue (577) within the motif is phosphorylated via Jak2 and associates with proliferative function. We investigated the phosphorylation status of the beta chain motif in response to GM-CSF stimulation in blasts cells from 12 patients with AML, and in blood monocytes from 6 healthy volunteers. Leukemic samples consisted of >99% blasts after buffy coat separation according to cytospin morphology. All leukemic blasts and blood monocyte samples expressed alpha and beta chain by flow cytometry. The phosphorylation status of residues 577 and 585 were examined utilising beta chain phospho-specific antibodies, after stimulation of AML blasts or blood monocytes with GM-CSF and beta chain immunoprecipitation. Human monocyte preparations all showed inducible serine 585 phosphorylation and tyrosine 577 phosphorylation. In contrast AML blasts displayed strong constitutive serine 585 phosphorylation in 10 out of 12 patients. No discernible tyrosine 577 phosphorylation in the absence of cytokine was observed for the AML samples. Beta chain phosphorylation pattern was independent of FAB classification. Inhibition of Jak2 did not prevent serine 585 phosphorylation of the receptor. Constitutive Akt activation was observed in AML blasts. The data provides evidence for dysregulation of a novel GM-CSF/IL-3 receptor mediated survival pathway involving serine/threonine kinases, 14-3-3 and Akt that is active in primary AML blasts in the majority of patients. This data would suggest that dysregulated activation of serine/threonine kinases and/or phosphatases at the receptor level may contribute to leukemogenesis.

scholarly journals The Meningococcal Adhesin NhhA Provokes Proinflammatory Responses in Macrophages via Toll-Like Receptor 4-Dependent and -Independent Pathways

2012 ◽  
Vol 80 (11) ◽  
pp. 4027-4033 ◽  
Author(s):  
Mikael Sjölinder ◽  
Georg Altenbacher ◽  
Xiao Wang ◽  
Yumin Gao ◽  
Charlotta Hansson ◽  
...  
Keyword(s):  
Gene Regulation ◽  
Gene Activation ◽  
Adaptor Protein ◽  
Human Monocyte ◽  
Colony Stimulating Factor ◽  
Gm Csf ◽  
Proinflammatory Responses ◽  
Macrophage Colony ◽  
Related Proteins ◽  
Stimulating Factor

ABSTRACTActivation of macrophages by Toll-like receptors (TLRs) and functionally related proteins is essential for host defense and innate immunity. TLRs recognize a wide variety of pathogen-associated molecules. Here, we demonstrate that the meningococcal outer membrane protein NhhA has immunostimulatory functions and triggers release of proinflammatory cytokines from macrophages. NhhA-induced cytokine release was found to proceed via two distinct pathways in RAW 264.7 macrophages. Interleukin-6 (IL-6) secretion was dependent on activation of TLR4 and required the TLR signaling adaptor protein MyD88. In contrast, release of tumor necrosis factor (TNF) was TLR4 and MyD88 independent. Both pathways involved NF-κB-dependent gene regulation. Using a PCR-based screen, we could identify additional targets of NhhA-dependent gene activation such as the cytokines and growth factors IL-1α, IL-1β, granulocyte colony-stimulating factor (G-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF). In human monocyte-derived macrophages, G-CSF, GM-CSF, and IL-6 were found to be major targets of NhhA-dependent gene regulation. NhhA induced transcription of IL-6 and G-CSF mRNA via TLR4-dependent pathways, whereas GM-CSF transcription was induced via TLR4-independent pathways. These data provide new insights into the role of NhhA in host-pathogen interaction.

scholarly journals Monoclonal Antibodies Capable of Binding SARS-CoV-2 Spike Protein Receptor Binding Motif Specifically Prevent GM-CSF Induction

2020 ◽  
Author(s):  
Xiaoling Qiang ◽  
Shu Zhu ◽  
Jianhua Li ◽  
Ping Wang ◽  
Kevin J. Tracey ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Receptor Binding ◽  
Viral Entry ◽  
Protein Targeting ◽  
Inflammatory Responses ◽  
Human Monocyte ◽  
Spike Protein ◽  
Binding Motif ◽  
Gm Csf ◽  
Future Assessment

AbstractA severe acute respiratory syndrome (SARS)-like coronavirus (SARS-CoV-2) has recently caused a pandemic COVID-19 disease that infected more than 25.6 million and killed 852,000 people worldwide. Like the SARS-CoV, SARS-CoV-2 also employs a receptor-binding motif (RBM) of its envelope spike protein for binding the host angiotensin-converting enzyme 2 (ACE2) to gain viral entry. Currently, extensive efforts are being made to produce vaccines against a surface fragment of a SARS-CoV-2, such as the spike protein, in order to boost protective antibody responses. It was previously unknown how spike protein-targeting antibodies would affect innate inflammatory responses to SARS-CoV-2 infections. Here we generated a highly purified recombinant protein corresponding to the RBM of SARS-CoV-2, and used it to screen for cross-reactive monoclonal antibodies (mAbs). We found two RBM-binding mAbs that competitively inhibited its interaction with human ACE2, and specifically blocked the RBM-induced GM-CSF secretion in both human monocyte and murine macrophage cultures. Our findings have suggested a possible strategy to prevent SARS-CoV-2-elicited “cytokine storm”, and provided a potentially useful criteria for future assessment of innate immune-modulating properties of various SARS-CoV-2 vaccines.One Sentence SummaryRBM-binding Antibodies Inhibit GM-CSF Induction.

Serial Analysis of Gene Expression in Human Monocyte-Derived Dendritic Cells

Blood ◽  
1999 ◽  
Vol 94 (3) ◽  
pp. 845-852 ◽  
Author(s):  
Shin-ichi Hashimoto ◽  
Takuji Suzuki ◽  
Hong-Yan Dong ◽  
Shigenori Nagai ◽  
Nobuyuki Yamazaki ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dendritic Cells ◽  
Cell Structure ◽  
Human Monocyte ◽  
Interleukin 4 ◽  
Maturation Stage ◽  
Blood Monocytes ◽  
Gm Csf ◽  
Highly Expressed Genes

Dendritic cells (DCs) are professional antigen-presenting cells in the immune system and can be generated in vitro from hematopoietic progenitor cells in the bone marrow, CD34+ cord blood cells, precursor cells in the peripheral blood, and blood monocytes by culturing with granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-4, and tumor necrosis factor-. We have performed serial analysis of gene expression (SAGE) in DCs derived from human blood monocytes. A total of 58,540 tag sequences from a DC complementary DNA (cDNA) library represented more than 17,000 different genes, and these data were compared with SAGE analysis of tags from monocytes (Mo) and GM-CSF–induced macrophages (M◊). Many of the genes that were differentially expressed in DCs were identified as genes encoding proteins related to cell structure and cell motility. Interestingly, the highly expressed genes in DCs encode chemokines such as TARC, MDC, and MCP-4, which preferentially chemoattract Th2-type lymphocytes. Although DCs have been considered to be very heterogeneous, the identification of specific genes expressed in human Mo-derived DCs should provide candidate genes to define subsets of, the function of, and the maturation stage of DCs and possibly also to diagnose diseases in which DCs play a significant role, such as autoimmune diseases and neoplasms. This study represents the first extensive gene expression analysis in any type of DCs.

scholarly journals Hematopoietic cytokines mediate resistance to targeted therapy in FLT3-ITD acute myeloid leukemia

2019 ◽  
Vol 3 (7) ◽  
pp. 1061-1072 ◽  
Author(s):  
Pamela J. Sung ◽  
Mayumi Sugita ◽  
Holly Koblish ◽  
Alexander E. Perl ◽  
Martin Carroll
Keyword(s):  
Acute Myeloid Leukemia ◽  
Cell Survival ◽  
Signaling Pathways ◽  
Myeloid Leukemia ◽  
Integration Site ◽  
Clinical Activity ◽  
Gm Csf ◽  
Flt3 Inhibitors ◽  
Acute Myeloid

Abstract Activating mutations in Fms-like tyrosine kinase 3 (FLT3) occur in ∼30% of adult cases of acute myeloid leukemia (AML). Selective second- and third-generation FLT3 inhibitors have shown significant clinical activity in patients with relapsed FLT3-mutant AML. However, clearance of FLT3-mutant clones does not consistently occur, and disease will progress in most patients after an initial response. This scenario challenges the model of FLT3-mutant AML being oncogene addicted, and it suggests that redundant signaling pathways regulate AML cell survival after FLT3 inhibition. We show that primary FLT3-mutant AML cells escape apoptosis induced by FLT3 inhibition in vitro in the presence of cytokines produced normally in the bone marrow, particularly granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3). Despite reactivating canonical FLT3-signaling pathways, GM-CSF and IL-3 maintain cell survival without rescuing proliferation. Cytokine-mediated resistance through GM-CSF and IL-3 is dependent on JAK kinase, STAT5, and proviral integration site of Moloney murine leukemia virus (PIM) but not MAPK or mammalian target of rapamycin signaling. Cotreatment with FLT3 inhibitors and inhibitors of JAK or PIM kinases blocks GM-CSF and IL-3 rescue of cell survival in vitro and in vivo. Altogether, these data provide a strong rationale for combination therapy with FLT3 inhibitors to potentially improve clinical responses in AML.

CCL19 and CCL21 Chemokines Induce Endocytosis and Augment Antigen Presentation in Human Mature Dendritic Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2651-2651
Author(s):  
Masahiro Ogasawara ◽  
Junji Tanaka ◽  
Masahiro Imamura ◽  
Masaharu Kasai
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
Antigen Presentation ◽  
Cd4 T Cells ◽  
Leukemia Cell ◽  
Human Monocyte ◽  
Blood Monocytes ◽  
Gm Csf ◽  
Cell Lysate ◽  
Cytokines And Chemokines

Abstract Dendritic cells (DCs) are potent antigen presenting cells capable of regulating immune responses. DCs lose the ability to capture and process antigens during maturation. In the present study, we examined the effects of CCR7 ligands, CCL19 and CCL21, on endocytosis and antigen presentation in human mature dendritic cells. Immature DCs were generated from peripheral blood monocytes by culturing with GM-CSF and IL4 for 2–3 days. For maturation, immature DCs were cultured with the addition of TNFα, IL1β, IL6 and prostaglandin E2 for another 24 hours. Immature or mature DCs were incubated with FITC-dextran with or without CCL19. Immature DCs internalized FITC-dextran efficiently independent of the presence of CCL19 after 1 hour incubation. On the other hand, mature DCs scarcely internalized FITC-dextran without CCL19. In the presence of CCL19, however, mature DCs internalized FITC-dextran significantly (approximately 60% positive). The effect of CCL19 on the uptake of FITC-dextran in mature DCs was dose and time dependent. CCL21 exerted a similar effect on mature DCs. Next, we examined whether CCL19 facilitates antigen presentation in mature dendritic cells. CD4+ T cells were cultured with irradiated autologous mature DCs which had been incubated with leukemia cell lysate with or without CCL19. Marked proliferation of CD4+ T cells occurred only when these cells were cultured with mature DCs loaded with leukemia cell lysate in the presence of CCL19. This is the first demonstration that chemokines have a pivotal role in endocytosis and antigen presentation by human monocyte-derived dendritic cells to the best of our knowledge. These results demonstrated that generation of potent antigen-loaded mature DCs in relatively short term culture using various cytokines and chemokines may have an important clinical implication to facilitate DC-based immunotherapy.

Ultrastructural observations of human monocytes infected with HIV-1

1991 ◽  
Vol 49 ◽  
pp. 108-109
Author(s):  
James K. Koehler ◽  
Steven G. Reed ◽  
Joao S. Silva
Keyword(s):  
Gradient Centrifugation ◽  
Virus Production ◽  
Human Monocyte ◽  
Red Cross ◽  
Human Monocytes ◽  
Blood Monocytes ◽  
Hiv Replication ◽  
Electron Microscopic ◽  
Ultrastructural Studies ◽  
Hiv 1

As part of a larger study involving the co-infection of human monocyte cultures with HIV and protozoan parasites, electron microscopic observations were made on the course of HIV replication and infection in these cells. Although several ultrastructural studies of the cytopathology associated with HIV infection have appeared, few studies have shown the details of virus production in “normal,” human monocytes/macrophages, one of the natural targets of the virus, and suspected of being a locus of quiescent virus during its long latent period. In this report, we detail some of the interactions of developing virons with the membranes and organelles of the monocyte host.Peripheral blood monocytes were prepared from buffy coats (Portland Red Cross) by Percoll gradient centrifugation, followed by adherence to cover slips. 90-95% pure monocytes were cultured in RPMI with 5% non-activated human AB serum for four days and infected with 100 TCID50/ml of HIV-1 for four hours, washed and incubated in fresh medium for 14 days.

Ultrastructure of rat alveolar macrophages activated in situ by poly:IC

1987 ◽  
Vol 45 ◽  
pp. 768-769
Author(s):  
A. M. Klinkner ◽  
R. A. Weiss ◽  
A. Kelley ◽  
P. J. Bugelski
Keyword(s):  
Venous Blood ◽  
Intratracheal Instillation ◽  
Buffy Coat ◽  
Fischer 344 Rats ◽  
Blood Monocytes ◽  
Fischer 344 ◽  
Polyinosinic:Polycytidylic Acid ◽  
Macrophage Activator ◽  
Endogenous Peroxidase

Polyinosinic:polycytidylic acid is an inducer of interferon and a macrophage activator. We have found that intratracheal instillation of polyI:C (IT-pI:C) activates rat bronchoalveolar lavage cells (BAL) for a variety of functions. Examination of Giemsa stained, cytocentrifuge preparations showed that IT-pI:C induced a population of BAL not seen in resident BAL. The morphology of these cells suggested that they might be derived from blood monocytes. To test this hypothesis we have examined several populations of macrophages that had been stained for endogenous peroxidase activity as a marker of cells derived from the monocyte-macrophage lineage.Macrophages were obtained from Fischer 344 rats. Peritoneal exudate cells (PEC) were collected by lavage 4 days after i.p. injection of 20 ml 3% thioglycolate. Buffy coat monocytes were separated from venous blood from naive rats.

scholarly journals A critical cytoplasmic domain of the interleukin-5 (IL-5) receptor alpha chain and its function in IL-5-mediated growth signal transduction.

1994 ◽  
Vol 14 (11) ◽  
pp. 7404-7413 ◽  
Author(s):  
S Takaki ◽  
H Kanazawa ◽  
M Shiiba ◽  
K Takatsu
Keyword(s):  
Signal Transduction ◽  
Protein Tyrosine Kinase ◽  
Cytoplasmic Domain ◽  
Beta Chain ◽  
Cytoplasmic Domains ◽  
Interleukin 5 ◽  
Alpha Chain ◽  
Gm Csf ◽  
Response Expression ◽  
And Function

Interleukin-5 (IL-5) regulates the production and function of B cells, eosinophils, and basophils. The IL-5 receptor (IL-5R) consists of two distinct membrane proteins, alpha and beta. The alpha chain (IL-5R alpha) is specific to IL-5. The beta chain is the common beta chain (beta c) of receptors for IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF). The cytoplasmic domains of both alpha and beta chains are essential for signal transduction. In this study, we generated cDNAs of IL-5R alpha having various mutations in their cytoplasmic domains and examined the function of these mutants by expressing them in IL-3-dependent FDC-P1 cells. The membrane-proximal proline-rich sequence of the cytoplasmic domain of IL-5R alpha, which is conserved among the alpha chains of IL-5R, IL-3R, and GM-CSF receptor (GM-CSFR), was found to be essential for the IL-5-induced proliferative response, expression of nuclear proto-oncogenes such as c-jun, c-fos, and c-myc, and tyrosine phosphorylation of cellular proteins including JAK2 protein-tyrosine kinase. In addition, analysis using chimeric receptors which consist of the extracellular domain of IL-5R alpha and the cytoplasmic domain of beta c suggested that dimerization of the cytoplasmic domain of beta c may be an important step in activating the IL-5R complex and transducing intracellular growth signals.

scholarly journals Claudin-Low Breast Cancer Inflammatory Signatures Support Polarization of M1-Like Macrophages with Protumoral Activity

Cancers ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 2248
Author(s):  
Mayra Cecilia Suárez-Arriaga ◽  
Alfonso Méndez-Tenorio ◽  
Vadim Pérez-Koldenkova ◽  
Ezequiel M. Fuentes-Pananá
Keyword(s):  
Breast Cancer ◽  
Overall Survival ◽  
Triple Negative ◽  
Tumor Subtype ◽  
Blood Monocytes ◽  
Adverse Clinical Outcome ◽  
M1 Macrophages ◽  
Peripheral Blood Monocytes ◽  
Gm Csf ◽  
Th1 Immune Response

We previously reported that triple-negative breast cancer (BRCA) cells overexpress the cytokines GM-CSF, G-CSF, MCP-1, and RANTES, and when monocytes were 3-D co-cultured with them, M1-like macrophages were generated with the ability to induce aggressive features in luminal BRCA cell lines. These include upregulation of mesenchymal and stemness markers and invasion. In this study, we stimulated peripheral blood monocytes with the four cytokines and confirmed their capacity to generate protumoral M1-like macrophages. Using the METABRIC BRCA database, we observed that GM-CSF, MCP-1, and RANTES are associated with triple-negative BRCA and reduced overall survival, particularly in patients under 55 years of age. We propose an extended M1-like macrophage proinflammatory signature connected with these three cytokines. We found that the extended M1-like macrophage signature coexists with monocyte/macrophage, Th1 immune response, and immunosuppressive signatures, and all are enriched in claudin-low BRCA samples, and correlate with reduced patient overall survival. Furthermore, we observed that all these signatures are also present in mesenchymal carcinomas of the colon (COAD) and bladder (BLCA). The claudin-low tumor subtype has an adverse clinical outcome and remains poorly understood. This study places M1 macrophages as potential protumoral drivers in already established cancers, and as potential contributors to claudin-low aggressiveness and poor prognosis.

scholarly journals Cytokines in chronic inflammatory arthritis. IV. Granulocyte/macrophage colony-stimulating factor-mediated induction of class II MHC antigen on human monocytes: a possible role in rheumatoid arthritis.

1989 ◽  
Vol 170 (3) ◽  
pp. 865-875 ◽  
Author(s):  
J M Alvaro-Gracia ◽  
N J Zvaifler ◽  
G S Firestein
Keyword(s):  
Rheumatoid Arthritis ◽  
Class Ii ◽  
Tnf Alpha ◽  
Blood Monocytes ◽  
Ifn Gamma ◽  
Normal Peripheral Blood ◽  
Gm Csf ◽  
Hla Dr ◽  
Class Ii Mhc ◽  
Hla Dq

Granulocyte/macrophage CSF (GM-CSF) has recently been identified in rheumatoid arthritis (RA) synovial effusions. To study a potential role for GM-CSF and other cytokines on the induction of HLA-DR expression on monocytes and synovial macrophages, we analyzed the relative ability of recombinant human cytokines to induce the surface expression of class II MHC antigens on normal peripheral blood monocytes by FACS analysis. GM-CSF (800 U/ml) (mean fluorescence channel 2.54 +/- 0.33 times the control, p less than 0.001) and IFN-gamma (100 U/ml) (5.14 +/- 0.60, p less than 0.001) were the most potent inducers of HLA-DR. TNF-alpha and IL-4 also increased HLA-DR expression, although to a lesser degree [1.31 +/- 0.06 (p less than 0.02) and 1.20 +/- 0.03 (p less than 0.01), respectively]. IL-1 (40 U/ml), IL-2 (10 ng/ml), IL-3 (50 U/ml), IL-6 (100 U/ml), and CSF-1 (1,000 U/ml) did not affect surface HLA-DR density. GM-CSF also increased HLA-DR mRNA expression and surface HLA-DQ expression, but decreased CD14 (a monocyte/macrophage antigen) expression. The effect of GM-CSF on HLA-DR was not mediated by the generation of IFN-gamma in vitro because it was not blocked by anti-IFN-gamma mAb. GM-CSF was additive with IL-4 and low amounts (less than 3 U/ml) of IFN-gamma and synergistic with TNF-alpha. Because we have recently reported that supernatants of cultured RA synovial cells produce a non-IFN-gamma factor that induces HLA-DR on monocytes, we then attempted to neutralize this factor with specific anti-GM-CSF mAb. Four separate synovial tissue supernatants were studied, and the antibody neutralized the HLA-DR-inducing factor in each (p less than 0.01).

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