scholarly journals Mature human eosinophils express functional Notch ligands mediating eosinophil autocrine regulation

Blood ◽  
2009 ◽  
Vol 113 (13) ◽  
pp. 3092-3101 ◽  
Author(s):  
Amy L. Radke ◽  
Lauren E. Reynolds ◽  
Rossana C. N. Melo ◽  
Ann M. Dvorak ◽  
Peter F. Weller ◽  
...  
Keyword(s):  
Notch Signaling ◽  
Neutralizing Antibodies ◽  
Receptor Activation ◽  
Notch Receptor ◽  
Gm Csf ◽  
Tissue Eosinophilia ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Blood Eosinophils ◽  
Notch Ligands

Abstract Eosinophil chemotaxis and survival within tissues are key components in the development of tissue eosinophilia and subsequent effector responses. In this study, we demonstrate a novel mechanism of eosinophil autoregulation affecting migration and survival mediated through Notch signaling. We show for the first time that human blood eosinophils express Notch receptors and Notch ligands, expressions of which are influenced by the presence of eosinophil-activating granulocyte-macrophage colony-stimulating factor (GM-CSF). Evidence of Notch receptor activation and subsequent transcription of the Notch-responsive gene HES1 were observed in GM-CSF–stimulated eosinophils, confirming functionality of eosinophil-expressed Notch-signaling components. Moreover, by inhibiting Notch signaling with γ-secretase inhibitors or Notch receptor–specific neutralizing antibodies, we demonstrate that autocrine Notch signaling enhances stimulus-mediated actin rearrangement and eosinophil chemokinesis, and impairs eosinophil viability. Taken together, these data suggest autocrine Notch signaling, enhanced in response to tissue- or inflammatory-derived signals, influences eosinophil activity and longevity, which may ultimately contribute to the development of tissue eosinophilia and exacerbation or remediation of eosinophil effector functions.

scholarly journals Identification of functionally distinct domains of human granulocyte- macrophage colony-stimulating factor using monoclonal antibodies

Blood ◽  
1991 ◽  
Vol 77 (5) ◽  
pp. 1033-1043 ◽  
Author(s):  
Y Kanakura ◽  
SA Cannistra ◽  
CB Brown ◽  
M Nakamura ◽  
GF Seelig ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Neutralizing Antibodies ◽  
Solid Phase ◽  
Receptor Interaction ◽  
Enzyme Linked Immunosorbent Assay ◽  
Colony Stimulating Factor ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Abstract Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a glycoprotein that is required for the survival, growth, and differentiation of hematopoietic progenitor cells. Although the primary structure of GM-CSF is known from cDNA cloning, the relationship between structure and function of GM-CSF is not fully understood. Fifteen different monoclonal antibodies (MoAbs) to human GM-CSF were generated to map immunologically distinct areas of the molecule. Each of the MoAbs was biotinylated and shown by enzyme-linked immunosorbent assay to bind to recombinant GM-CSF that had been affixed to a solid phase. Each of the 15 unconjugated MoAbs was then used to compete with each biotinylated MoAb for binding to GM-CSF. These cross-blocking studies identified eight distinct epitopes of native GM-CSF. Seven of these epitopes were also present in denatured GM-CSF by Western blotting, and four of the epitopes were at least partially conserved on GM-CSF that was reduced in beta-mercaptoethanol. MoAbs to four of eight epitopes neutralized both recombinant (glycosylated and nonglycosylated) and natural human GM-CSF in a GM colony-forming unit (CFU-GM) assay and blocked GM-CSF-induced activation of neutrophils. For most of the antibodies there was a good correlation between neutralizing activity and the capacity to block binding of 125I-GM-CSF to neutrophils or blasts. Non-neutralizing antibodies to one epitope partially blocked binding of 125I-GM-CSF to neutrophils. None of the MoAbs neutralized interleukin-3, G-CSF, or M-CSF. The locations of seven of the epitopes could be partially mapped with regard to the amino acid structure by determining reactivity to GM-CSF synthetic peptides or to human-mouse chimeric GM-CSFs. The neutralizing antibodies were found to map to amino acids 40–77, 78–94, or 110–127. Thus, these MoAbs are useful to identify functional domains of GM-CSF and in identifying regions that are likely to be involved in receptor interaction.

scholarly journals Further examination of the effects of recombinant cytokines on the proliferation of human megakaryocyte progenitor cells

Blood ◽  
1991 ◽  
Vol 77 (11) ◽  
pp. 2339-2346 ◽  
Author(s):  
E Bruno ◽  
RJ Cooper ◽  
RA Briddell ◽  
R Hoffman
Keyword(s):  
Colony Formation ◽  
Neutralizing Antibodies ◽  
De Novo ◽  
Fibrin Clot ◽  
Gm Csf ◽  
Normal Human ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Effector Pathways ◽  
Normal Human Bone Marrow

Abstract The effect of several recombinant cytokines, including interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), IL- 6, and IL-1 alpha, on megakaryocyte (MK) colony formation by a normal human bone marrow subpopulation (CD34+ DR+), enriched for the MK colony- forming unit (CFU-MK), was studied using a serum-depleted, fibrin clot culture system. IL-3 and GM-CSF, but not IL-6 or IL-1 alpha, stimulated MK colony formation by CD34+ DR+ cells. However, the addition of IL-1 alpha to CD34+ DR+ cultures containing IL-6 resulted in the appearance of CFU-MK-derived colonies, suggesting that IL-6 requires the presence of IL-1 alpha to exhibit its MK colony-stimulating activity (MK-CSA). Addition of neutralizing antibodies to IL-3 and GM-CSF, but not to IL-6 and IL-1 alpha, specifically inhibited the MK-CSA of IL-3 and GM-CSF, respectively. The addition of either anti-IL-6, anti-IL-1 alpha, or anti-IL-3 antisera to cultures containing both IL-6 and IL-1 alpha totally abolished the MK-CSA of the IL-6/IL-1 alpha combination. However, neither anti-IL-3 nor anti-GM-CSF antisera could totally neutralize the additive effect of the combination of IL-3 and GM-CSF on MK colony formation, indicating that these two cytokines act by affecting distinct effector pathways. These results suggest that while IL-3 and GM-CSF can directly affect CFU-MK-derived colony formation, IL- 1 alpha and IL-6 act in concert to promote de novo elaboration of IL-3 and thereby promote CFU-MK proliferative capacity.

Role for Bcl-xL in Delayed Eosinophil Apoptosis Mediated by Granulocyte-Macrophage Colony-Stimulating Factor and Interleukin-5

Blood ◽  
1998 ◽  
Vol 92 (3) ◽  
pp. 778-783 ◽  
Author(s):  
Birgit Dibbert ◽  
Isabelle Daigle ◽  
Doris Braun ◽  
Corinna Schranz ◽  
Martina Weber ◽  
...  
Keyword(s):  
Inflammatory Cells ◽  
Colony Stimulating Factor ◽  
Interleukin 5 ◽  
Gm Csf ◽  
Protein Levels ◽  
Tissue Eosinophilia ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor ◽  
Eosinophil Apoptosis

Eosinophils are potent inflammatory cells involved in allergic reactions. Inhibition of apoptosis of purified eosinophils by certain cytokines has been previously shown to be an important mechanism causing tissue eosinophilia. To elucidate the role of Bcl-2 family members in the inhibition of eosinophil apoptosis, we examined the expression of the known anti-apoptotic genes Bcl-2, Bcl-xL, and A1, as well as Bax and Bcl-xS, which promote apoptosis in other systems. We show herein that freshly isolated human eosinophils express significant amounts of Bcl-xL and Bax, but only little or no Bcl-2, Bcl-xS, or A1. As assessed by reverse transcription-polymerase chain reaction, immunoblotting, flow cytometry, and immunocytochemistry, we show that spontaneous eosinophil apoptosis is associated with a decrease in Bcl-xL mRNA and protein levels. In contrast, stimulation of the cells with granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-5 (IL-5) results in maintenance or upregulation of Bcl-xL mRNA and protein levels. Moreover, Bcl-2 protein is not induced by GM-CSF or IL-5 in purified eosinophils. Bcl-2 protein is also not expressed in tissue eosinophils as assessed by immunohistochemistry using two different eosinophilic tissue models. Furthermore, Bcl-xL antisense but not scrambled phosphorothioate oligodeoxynucleotides can partially block the cytokine-mediated rescue of apoptotic death in these cells. These data suggest that Bcl-xL acts as an anti-apoptotic molecule in eosinophils. © 1998 by The American Society of Hematology.

The Ig-like domain of human GM-CSF receptor α plays a critical role in cytokine binding and receptor activation

2010 ◽  
Vol 426 (3) ◽  
pp. 307-317 ◽  
Author(s):  
Shamaruh Mirza ◽  
Andrew Walker ◽  
Jinglong Chen ◽  
James M. Murphy ◽  
Ian G. Young
Keyword(s):  
Structural Alignment ◽  
Critical Role ◽  
Receptor Activation ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Subsequent Activation ◽  
Macrophage Colony ◽  
Key Residues ◽  
Stimulating Factor

GM-CSF (granulocyte/macrophage colony-stimulating factor) is an important mediator of inducible haemopoiesis and inflammation, and has a critical role in the function of alveolar macrophages. Its clinical applications include the mobilization of haemopoietic progenitors, and a role as an immune stimulant and vaccine adjuvant in cancer patients. GM-CSF signals via a specific α receptor (GM-CSFRα) and the shared hβc (human common β-subunit). The present study has investigated the role of the Ig-like domain of GM-CSFRα in GM-CSF binding and signalling. Deletion of the Ig-like domain abolished direct GM-CSF binding and decreased growth signalling in the presence of hβc. To locate the specific residues in the Ig-like domain of GM-CSFRα involved in GM-CSF binding, a structural alignment was made with a related receptor, IL-13Rα1 (interleukin-13 receptor α1), whose structure and mode of interaction with its ligand has recently been elucidated. Mutagenesis of candidate residues in the predicted region of interaction identified Val51 and Cys60 as having critical roles in binding to the α receptor, with Arg54 and Leu55 also being important. High-affinity binding in the presence of hβc was strongly affected by mutation of Cys60 and was also reduced by mutation of Val51, Arg54 and Leu55. Of the four key residues, growth signalling was most severely affected by mutation of Cys60. The results indicate a previously unrecognized role for the Ig-like domain, and in particular Cys60, of GM-CSFRα in the binding of GM-CSF and subsequent activation of cellular signalling.

scholarly journals Coordinate secretion of interleukin-1 beta and granulocyte-macrophage colony-stimulating factor by the blast cells of acute myeloblastic leukemia: role of interleukin-1 as an endogenous inducer

Blood ◽  
1990 ◽  
Vol 76 (8) ◽  
pp. 1481-1489 ◽  
Author(s):  
JC Rodriguez-Cimadevilla ◽  
V Beauchemin ◽  
L Villeneuve ◽  
F Letendre ◽  
A Shaw ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Interleukin 1 ◽  
Acute Myeloblastic Leukemia ◽  
Colony Stimulating Factor ◽  
Autocrine Growth ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
High Production ◽  
Macrophage Colony ◽  
Stimulating Factor

Abstract Acute myeloblastic leukemia (AML) blasts have been shown to produce a variety of cytokines in culture such as interleukin-1 (IL-1), IL-6, granulocyte-, macrophage-, and granulocyte-macrophage colony- stimulating factor (GM-CSF), and tumor necrosis factor-alpha (TNF alpha). Using two sensitive and specific enzyme-linked immunosorbent assays for IL-1 beta and GM-CSF, we document in the present study that the production of the two cytokines by AML blasts in culture is coordinated. First, we observe a striking correlation between the levels of GM-CSF and IL-1 beta released by the cells. Thus, a high production of IL-1 beta is always concordant with a high production of GM-CSF and, conversely, low production of IL-1 beta is concordant with low levels of GM-CSF. Second, neutralization of intrinsic IL-1 using antibodies that are specific for IL-1 alpha and -1 beta suppresses the release of GM-CSF by the cells. Third, neutralization of the endogenous source of IL-1 also results in an abrogation of GM-CSF mRNA. Fourth, the production of both IL-1 beta and GM-CSF is up-regulated by exposing AML blasts to an exogenous source of IL-1, suggesting a positive regulation of autocrine growth factor production. Taken together, our results indicate that GM-CSF production by AML blasts is mediated by endogenously produced IL-1. Both IL-1 beta and -1 alpha are produced by AML blasts, although IL-1 beta appears to be more abundant. Spontaneous colony formation by AML blasts is abrogated by the addition of neutralizing antibodies against IL-1 beta and GM-CSF, whereas each antibody alone has little effect on blast proliferation. Taken together, our results are consistent with the view that the production of IL-1 beta by AML blasts supports autocrine growth in culture, through induction of CSFs or other cytokines that stimulate blast proliferation.

Extracellular signal-regulated kinase mediates granulocyte-macrophage colony-stimulating factor messenger RNA stabilization in tumor necrosis factor-α plus fibronectin-activated peripheral blood eosinophils

Blood ◽  
2002 ◽  
Vol 99 (11) ◽  
pp. 4048-4052 ◽  
Author(s):  
Stéphane Esnault ◽  
James S. Malter
Keyword(s):  
Messenger Rna ◽  
Mrna Stabilization ◽  
Extracellular Signal Regulated Kinase ◽  
Gm Csf ◽  
Extracellular Signal ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Factor Α ◽  
Blood Eosinophils ◽  
Necrosis Factor

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is critical for promoting the long-term survival of lung- or airway-based eosinophils. Previously, we have shown that fibronectin and tumor necrosis factor α induced autocrine production of GM-CSF that markedly enhanced eosinophil survival. Cytokine release was preceded by and dependent on messenger RNA (mRNA) stabilization. Here, we show that mitogen-activated protein kinase (MAPK) activation is responsible for GM-CSF mRNA stabilization in peripheral blood eosinophils (pbeos). Activation of extracellular signal-regulated kinase (ERK) but not p38 correlated with GM-CSF mRNA stability. Although ERK inhibition completely prevented GM-CSF mRNA stabilization, p38 inhibition had a partial effect. To establish which MAPK was crucial, we transduced pbeos with dominant-active TatMEK1(E) or TatMKK3b(E) proteins that selectively phosphorylate ERK or p38, respectively. These studies showed that ERK but not p38 was sufficient for GM-CSF mRNA stabilization. These data are in contradistinction to the c-Jun NH2-termainal kinase–mediated regulation of interleukin 2 and 3 mRNAs and suggest unique regulatory features for GM-CSF mRNA in eosinophils.

scholarly journals Coordinate secretion of interleukin-1 beta and granulocyte-macrophage colony-stimulating factor by the blast cells of acute myeloblastic leukemia: role of interleukin-1 as an endogenous inducer

Blood ◽  
1990 ◽  
Vol 76 (8) ◽  
pp. 1481-1489 ◽  
Author(s):  
JC Rodriguez-Cimadevilla ◽  
V Beauchemin ◽  
L Villeneuve ◽  
F Letendre ◽  
A Shaw ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Interleukin 1 ◽  
Acute Myeloblastic Leukemia ◽  
Colony Stimulating Factor ◽  
Autocrine Growth ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
High Production ◽  
Macrophage Colony ◽  
Stimulating Factor

Acute myeloblastic leukemia (AML) blasts have been shown to produce a variety of cytokines in culture such as interleukin-1 (IL-1), IL-6, granulocyte-, macrophage-, and granulocyte-macrophage colony- stimulating factor (GM-CSF), and tumor necrosis factor-alpha (TNF alpha). Using two sensitive and specific enzyme-linked immunosorbent assays for IL-1 beta and GM-CSF, we document in the present study that the production of the two cytokines by AML blasts in culture is coordinated. First, we observe a striking correlation between the levels of GM-CSF and IL-1 beta released by the cells. Thus, a high production of IL-1 beta is always concordant with a high production of GM-CSF and, conversely, low production of IL-1 beta is concordant with low levels of GM-CSF. Second, neutralization of intrinsic IL-1 using antibodies that are specific for IL-1 alpha and -1 beta suppresses the release of GM-CSF by the cells. Third, neutralization of the endogenous source of IL-1 also results in an abrogation of GM-CSF mRNA. Fourth, the production of both IL-1 beta and GM-CSF is up-regulated by exposing AML blasts to an exogenous source of IL-1, suggesting a positive regulation of autocrine growth factor production. Taken together, our results indicate that GM-CSF production by AML blasts is mediated by endogenously produced IL-1. Both IL-1 beta and -1 alpha are produced by AML blasts, although IL-1 beta appears to be more abundant. Spontaneous colony formation by AML blasts is abrogated by the addition of neutralizing antibodies against IL-1 beta and GM-CSF, whereas each antibody alone has little effect on blast proliferation. Taken together, our results are consistent with the view that the production of IL-1 beta by AML blasts supports autocrine growth in culture, through induction of CSFs or other cytokines that stimulate blast proliferation.

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