Studies of the inhibitory activity of MK-0591 (3-[1-(4-chlorobenzyl)-3-(t-butylthio)-5-(quinolin-2-yl-methoxy)-indol-2-yl]-2,2-dimethyl propanoic acid) on arachidonic acid metabolism in human phagocytes

1992 ◽  
Vol 70 (6) ◽  
pp. 808-813 ◽  
Author(s):  
Luc Ménard ◽  
Michel Laviolette ◽  
Pierre Borgeat
Keyword(s):  
Arachidonic Acid ◽  
Inhibitory Activity ◽  
Platelet Activating Factor ◽  
Specific Inhibitor ◽  
Oxidation Products ◽  
Propanoic Acid ◽  
Ionophore A23187 ◽  
Colony Stimulating Factor ◽  
Trans Isomers ◽  
Stimulating Factor

We have investigated the inhibitory activity of compound MK-0591 (3-[1-(4-chlorobenzyl)-3-(t-butylthio)-5-(quinolin-2-yl-methoxy)-indol-2-yl]-2,2-dimethyl propanoic acid) on 5-lipoxygenase (5-LO) product synthesis in various human phagocytes stimulated with either the ionophore A23187, opsonized zymosan (OPZ), platelet-activating factor (PAF), or forrnyl-methionyl-leucyl-phenylalanine (fMLP). The lipoxygenase products were analyzed by reversed-phase HPLC. MK-0591 inhibited the formation of 5-hydroxyeicosatetraenoic acid, leukotriene (LT) B4, its Ω-oxidation products, and 6-trans-isomers with IC50 values of 2.8–4.8 nM in A23187-stimulated neutrophils. In these conditions, arachidonic acid at a concentration of 10 μM had no effect on MK-0591 inhibitory activity. In neutrophils stimulated with OPZ, the synthesis of LTB4, its Ω-oxidation products, and 6-trans-isomers was inhibited with IC50 values of 9.5–11.0 nM. MK-0591 inhibited 5-LO product synthesis in A23187-stimulated blood monocytes, eosinophils, and alveolar macrophages with IC50 values of 0.3–0.9, 3.7–5.3, and 8.5–17.3 nM, respectively. In neutrophils primed with granulocyte – macrophage colony-stimulating factor and stimulated with PAF, lipoxygenase product synthesis was inhibited with IC50 values of 7.7–8.7 nM. At the concentration of 1 μM, MK-0591 had no inhibitory effect on 15-lipoxygenase activity in human polymorphonuclear leukocytes, nor on human platelet 12-lipoxygenase and cyclooxygenase. In conclusion, MK-0591 is a very potent and specific inhibitor of 5-LO product synthesis in various types of human phagocytes.Key words: 5-lipoxygenase inhibition, colony-stimulating factor, leukotriene, neutrophil, eosinophil, monocyte, macrophage, platelet-activating factor.

scholarly journals Granulocyte-macrophage colony-stimulating factor can stimulate macrophage proliferation via persistent activation of Na+/H+ antiport. Evidence for two distinct roles for Na+/H+ antiport activation

1990 ◽  
Vol 265 (2) ◽  
pp. 359-364 ◽  
Author(s):  
S J Vallance ◽  
C P Downes ◽  
E J Cragoe ◽  
A D Whetton
Keyword(s):  
Intracellular Ph ◽  
Platelet Activating Factor ◽  
Colony Stimulating Factor ◽  
Gm Csf ◽  
Transduction Mechanisms ◽  
Cellular Signal ◽  
Extracellular Stimuli ◽  
Macrophage Colony ◽  
Continuous Presence ◽  
Stimulating Factor

Macrophages respond to a variety of extracellular stimuli which can modulate the proliferation, development, activation and functional activity of these cells. The effects of two such agents, granulocytemacrophage colony-stimulating factor (GM-CSF, which stimulates proliferation) and platelet-activating factor (PAF, which stimulates chemotaxis and bactericidal activity), on cellular signal transduction mechanisms were compared. PAF can stimulate inositol lipid hydrolysis leading to Ca2+ mobilization. GM-CSF on the other hand has no effect on these events. Both agonists do, however, share an ability to activate an amiloride-sensitive Na+/H+ antiport and, furthermore, amiloride analogues are shown to inhibit the proliferative effects of GM-CSF on these cells. Long-term incubations with either PAF or GM-CSF demonstrate that it is only those cells pretreated with the latter which show a persistent activation of the antiport together with a sustained increase in intracellular pH. PAF-treated cells exhibit only a transitory increase in antiport activity, their intracellular pH levels returning to resting levels in spite of the continuous presence of the agonist in the medium. These effects of GM-CSF and PAF on Na+/H+ exchange are observed in both bicarbonate-free and bicarbonate-containing medium. These results lead us to suggest that the Na+/H+ antiport has a role in macrophage proliferation and in the regulation of intracellular pH during the oxidative burst stimulated by PAF and other agonists, and that differential mechanisms whereby this antiport is regulated exist in macrophages.

scholarly journals Phosphorylation-dependent and -independent pathways of platelet aggregation

1989 ◽  
Vol 258 (2) ◽  
pp. 479-485 ◽  
Author(s):  
S P Watson ◽  
S Hambleton
Keyword(s):  
Protein Phosphorylation ◽  
Platelet Activating Factor ◽  
Specific Inhibitor ◽  
Human Platelets ◽  
Signalling Pathways ◽  
Ionophore A23187 ◽  
Phorbol Dibutyrate ◽  
Kinase C ◽  
Dependent Pathway

We have used the non-specific inhibitor of protein kinases, staurosporine, to investigate the role of protein phosphorylation during aggregation, the mobilization of intracellular Ca2+ (Ca2+)i and intracellular pH (pHi) in thrombin-stimulated platelets. The concentration of staurosporine chosen for these studies, 1 microM, was previously reported to inhibit protein phosphorylation completely but to have no effect on the activation of phospholipase C in thrombin-stimulated human platelets [Watson, McNally, Shipman & Godfrey (1988) Biochem. J. 249, 345-350]. Aggregation induced by phorbol dibutyrate is slow (several minutes) and is inhibited completely by staurosporine. In contrast, aggregation induced by thrombin, platelet-activating factor or ionophore A23187 is rapid (occurs within 60 s), and is slowed, but not inhibited, in the presence of staurosporine. On the other hand, staurosporine causes a small potentiation of the peak [Ca2+]i signal induced by thrombin and a marked increase in the half-life of decay of this signal, but has no effect on pHi. Under conditions designed to prevent an increase in [Ca2+]i (presence of Ni2+ to prevent Ca2+ entry, and depletion of the intracellular Ca2+ stores), aggregation induced by thrombin resembles that by phorbol dibutyrate and is now inhibited completely by staurosporine. Taken together, these results provide evidence for two signalling pathways for aggregation, a relatively rapid phosphorylation-independent route mediated by Ca2+ and a slower, phosphorylation-dependent, pathway mediated by protein kinase C. Since staurosporine slows aggregation induced by thrombin, it appears that under normal conditions these pathways interact synergistically.

scholarly journals Features of Macrophage Differentiation Induced by p19INK4d, a Specific Inhibitor of Cyclin D–Dependent Kinases

Blood ◽  
1997 ◽  
Vol 90 (1) ◽  
pp. 126-137 ◽  
Author(s):  
Masashi Adachi ◽  
Martine F. Roussel ◽  
Karin Havenith ◽  
Charles J. Sherr
Keyword(s):  
Kinase Activity ◽  
Specific Inhibitor ◽  
Mononuclear Phagocytes ◽  
G1 Phase ◽  
Cyclin D ◽  
Colony Stimulating Factor ◽  
Phase Arrest ◽  
G1 Phase Arrest ◽  
Metallothionein Promoter ◽  
Stimulating Factor

The mitogen-dependent induction of cyclin D–dependent kinase activity is required for cells to enter the DNA synthetic (S) phase of their division cycle. Immature 32Dcl3 myeloid cells (32D) proliferating in the presence of interleukin-3 (IL-3) normally express cyclins D2 and D3, which assemble into binary holoenzyme complexes with their catalytic subunits, CDK4 and CDK6. When 32D cells are switched to medium containing granulocyte colony-stimulating factor (G-CSF ) instead of IL-3, D-type cyclins are degraded and, in the absence of their associated kinase activity, the cells arrest in the first gap phase (G1 ) of the cell cycle and differentiate to neutrophils. We derived 32D cells in which the expression of p19INK4d, a specific polypeptide inhibitor of CDK4 and CDK6, is regulated by the heavy metal-inducible sheep metallothionein promoter. Induction of p19INK4d in response to zinc prolonged cell survival in the absence of growth factor treatment. When maintained in medium containing both IL-3 and zinc, these cells lost cyclin D–dependent kinase activity, underwent G1 phase arrest, and acquired certain morphologic, antigenic, and functional properties of mononuclear phagocytes. Cells induced to express p19INK4d did not synthesize receptors for macrophage colony-stimulating factor (M-CSF/CSF-1) and reverted to an immature myeloid phenotype when shifted back into medium containing IL-3 alone. These cells exhibited accelerated differentiation to neutrophils in response to G-CSF but also gave rise to macrophage-like cells when maintained in medium containing both G-CSF and zinc. Therefore, the acquisition of macrophage properties in response to zinc treatment neither depended upon IL-3 nor upon G1 phase arrest per se and instead reflects some ability of p19INK4d, and presumably cyclin D–dependent kinases, to affect myeloid differentiation.

scholarly journals Inhibition of hematopoiesis in normal human long-term marrow cultures treated with recombinant human macrophage colony-stimulating factor

Blood ◽  
1991 ◽  
Vol 78 (3) ◽  
pp. 651-657 ◽  
Author(s):  
H Mayani ◽  
LJ Guilbert ◽  
SC Clark ◽  
A Janowska-Wieczorek
Keyword(s):  
Inhibitory Activity ◽  
Inhibitory Effect ◽  
Human Macrophage ◽  
Colony Stimulating Factor ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor ◽  
Stromal Layer ◽  
Marrow Cultures

Abstract The effects of recombinant human macrophage colony-stimulating factor (rhCSF-1) in long-term marrow cultures (LTMC) established from normal bone marrow cells were examined. When added during the first 3 weeks of culture (every second day, at 15 ng/mL), rhCSF-1 strongly inhibited the growth of all hematopoietic progenitors analyzed (colony-forming unit- MIX [CFU-MIX], CFU-granulocyte macrophage [CFU-GM], CFU-M, CFU-G, burst- forming unit-erythroid). Paralleling the inhibition of progenitors was the complete loss of adipocytes from the stromal layer of rhCSF-1- treated cultures. The inhibitory effect of rhCSF-1 correlated in all instances with the accumulation in the supernatants of these cultures of an activity (different from CSF-1) that inhibited colony formation in semisolid cultures. When addition of rhCSF-1 was delayed 3 weeks, its inhibitory effects were significantly reduced, which correlated with reduced inhibitory activity detected in the supernatants. Analysis of CSF-1 concentration by radioreceptor assay confirmed that added rhCSF-1 increased culture CSF-1 levels and showed that the decreased inhibition observed when rhCSF-1 is added later in culture was not due to decreased CSF-1 levels at that point. In contrast, the ability of rhCSF-1 to inhibit hematopoiesis and accumulate inhibitory activity in LTMC correlated with its rate of utilization, much higher in the first 2 weeks of culture, when the stromal layer was being established, than later. These observations document the inhibitory effect of rhCSF-1 on all aspects of hematopoiesis conducted in cultures that simulate the hematopoietic microenvironment, demonstrate the importance of accessory/stromal cells in mediating the effects of rhCSF-1 in LTMC, and point to an inhibitory activity as the mediating agent.

scholarly journals Expression of mRNA and immunoreactivity for the granulocyte/macrophage colony-stimulating factor in activated human eosinophils.

1991 ◽  
Vol 174 (3) ◽  
pp. 749-752 ◽  
Author(s):  
R Moqbel ◽  
Q Hamid ◽  
S Ying ◽  
J Barkans ◽  
A Hartnell ◽  
...  
Keyword(s):  
Human Peripheral Blood ◽  
Allergic Inflammation ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Colony Stimulating Factor ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Using in situ hybridization, we have shown that activated human peripheral blood eosinophils express mRNA for granulocyte/macrophage colony-stimulating factor (GM-CSF). Between 15 and 27% of eosinophils gave positive hybridization signals for GM-CSF mRNA after stimulation with the calcium ionophore A23187 or interferon gamma, and 4 and 6% after incubation with interleukin 3 (IL-3) or IL-5. Activated eosinophils also gave specific immunoreactivity with an anti-GM-CSF polyclonal antibody, suggesting translation of the mRNA. These data indicate that eosinophils may be an important source of GM-CSF at sites of allergic inflammation. Furthermore, the identification of GM-CSF production by human eosinophils suggests that the pro-inflammatory potential of this cell type may be substantially greater than hitherto recognized.

scholarly journals Inhibition of Monocytic Interleukin-12 Production by Candida albicans via Selective Activation of ERK Mitogen-Activated Protein Kinase

2004 ◽  
Vol 72 (5) ◽  
pp. 2513-2520 ◽  
Author(s):  
Ningfeng Tang ◽  
Liming Liu ◽  
Kefei Kang ◽  
Pranab K. Mukherjee ◽  
Masakazu Takahara ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Protein Kinase ◽  
Inhibitory Activity ◽  
Biochemical Analysis ◽  
Specific Inhibitor ◽  
Mitogen Activated Protein Kinase ◽  
Interleukin 12 ◽  
Mitogen Activated Protein ◽  
Ifn Γ ◽  
Stimulating Factor

ABSTRACT Our previous data demonstrated that live Candida albicans inhibits interleukin-12 (IL-12) production by human monocytes. Here we explored whether C. albicans inhibits IL-12 via a released factor and whether the inhibition is mediated via mitogen-activated protein kinase (MAPK) regulation. Supernatant fluids were obtained from cultured C. albicans (SC5314) as well as cultured Saccharomyces cerevisiae after 20 h of incubation. At 2 h postincubation of monocytes with heat-killed C. albicans (HKCA) (2:1) to stimulate IL-12, concentrated fungal supernatant fluids were added and incubated for an additional 20 h. The present data show that, unlike supernatant fluids obtained from S. cerevisiae, the C. albicans supernatant fluids significantly suppressed IL-12 production induced by HKCA. This suggested that the inhibition is Candida specific. A preliminary biochemical analysis revealed that this secretory IL-12 inhibitory factor is glycoprotein in nature. The inhibitory activity had no effect on the phagocytosis of yeasts. Supernatant fluids from C. albicans markedly induced the phosphorylation of ERK44/42 MAPK, but not p38 and SAPK, 1 min after they were added to monocytes. To test if the induction of ERK44/42 MAPK was central to the IL-12 inhibition, we used gamma interferon (IFN-γ) (1 ng/ml) plus lipopolysaccharide (LPS) (100 ng/ml) to stimulate IL-12 production by monocytes. The inhibition of ERK MAPK by the specific inhibitor PD 98059 significantly reduced phospho-ERK44/42 MAPK levels induced by C. albicans supernatant fluids in the IFN-γ-plus-LPS-driven monocytes. Concomitantly, PD 98059 reversed the IL-12 inhibitory activity of the C. albicans supernatant (P < 0.01). These data indicate that C. albicans can inhibit IL-12 production by secreting an ERK44/42 MAPK-stimulating factor and thus can attenuate effective immune responses.

scholarly journals Calcineurin potentiates activation of the granulocyte-macrophage colony-stimulating factor gene in T cells: involvement of the conserved lymphokine element 0.

1994 ◽  
Vol 5 (1) ◽  
pp. 119-128 ◽  
Author(s):  
A Tsuboi ◽  
E S Masuda ◽  
Y Naito ◽  
H Tokumitsu ◽  
K Arai ◽  
...  
Keyword(s):  
T Cell ◽  
Binding Site ◽  
Calcium Ionophore ◽  
Regulatory Elements ◽  
Ionophore A23187 ◽  
Colony Stimulating Factor ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-2 (IL-2) are produced by stimulation with phorbol-12-myristate acetate (PMA) and calcium ionophore (A23187) in human T cell leukemia Jurkat cells. The expression of GM-CSF and IL-2 is inhibited by immunosuppressive drugs such as cyclosporin A (CsA) and FK506. Earlier studies on the IL-2 gene expression showed that overexpression of calcineurin (CN), a Ca2+/calmodulin-dependent protein phosphatase, can stimulate transcription from the IL-2 promoter through the NF-AT-binding site. In this study, we obtained evidence that transfection of the cDNAs for CN A (catalytic) and CN B (regulatory) subunits also augments transcription from the GM-CSF promoter and recovers the transcription inhibited by CsA. The constitutively active type of the CN A subunit, which lacks the auto-inhibitory and calmodulin-binding domains, acts in synergy with PMA to activate transcription from the GM-CSF promoter. We also found that the active CN partially replaces calcium ionophore in synergy with PMA to induce expression of endogenous GM-CSF and IL-2. By multimerizing the regulatory elements of the GM-CSF promoter, we found that one of the target sites for the CN action is the conserved lymphokine element 0 (CLE0), located at positions between -54 and -40. Mobility shift assays showed that the CLE0 sequence has an AP1-binding site and is associated with an NF-AT-like factor, termed NF-CLE0 gamma. NF-CLE0 gamma binding is induced by PMA/A23187 and is inhibited by treatment with CsA. These results suggest that CN is involved in the coordinated induction of the GM-CSF and IL-2 genes and that the CLE0 sequence of the GM-CSF gene is a functional analogue of the NF-AT-binding site in the IL-2 promoter, which mediates signals downstream of T cell activation.

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