scholarly journals Tyrosine phosphorylation of mitogen-activated protein kinases is necessary for activation of murine macrophages by natural and synthetic bacterial products.

1993 ◽  
Vol 177 (4) ◽  
pp. 1071-1077 ◽  
Author(s):  
Z Dong ◽  
X Qi ◽  
I J Fidler
Keyword(s):  
Tyrosine Phosphorylation ◽  
Map Kinases ◽  
Kinase Assay ◽  
Tnf Alpha ◽  
Necrosis Factor Alpha ◽  
Murine Macrophages ◽  
Alpha Production ◽  
Mitogen Activated Protein ◽  
Molecular Masses ◽  
Bacterial Products

The purpose of these studies was to determine the intracellular signal transduction pathways of bacterial products in murine macrophages from lipopolysaccharide (LPS)-responder C3H/HeN and LPS-nonresponder C3H/HeJ mice. Both LPS and synthetic lipopeptide CGP 31362 (LPP) induced production of tumor necrosis factor alpha (TNF-alpha) in C3H/HeN macrophages. In C3H/HeJ macrophages, however, TNF-alpha was induced only by incubation with LPP. Both LPS and LPP induced tyrosine phosphorylation on proteins with apparent molecular masses of 39, 41, and 45 kD (p35, p41, and p45) in C3H/HeN macrophages, whereas in C3H/HeJ macrophages, tyrosine phosphorylation was induced only by LPP. 20-h incubation with LPS or LPP downregulated TNF-alpha production/secretion and tyrosine phosphorylation in C3H/HeN macrophages induced by additional LPS or LPP. In C3H/HeJ macrophages, however, the downregulation of TNF-alpha production and tyrosine phosphorylation were observed only with LPP. Protein kinase assays, Western blotting analyses, phenyl-Sepharose chromatography, and immunocomplex kinase assay suggested that p45 and p39 were similar or identical to mitogen-activated protein (MAP) kinase 1 and 2, respectively. Pretreatment of macrophages with LPS or LPP did not change the amount of kinase proteins but inhibited the stimulation of kinase activity by the agents. These data suggest that MAP kinases are among target proteins involved in the transduction of LPS and LPP signals that lead to activation of murine macrophages to produce/secrete TNF.

scholarly journals Effects of granulocyte-macrophage colony-stimulating factor and tumour necrosis factor-α on tyrosine phosphorylation and activation of mitogen-activated protein kinases in human neutrophils

1995 ◽  
Vol 307 (1) ◽  
pp. 39-45 ◽  
Author(s):  
W H Waterman ◽  
R I Sha'afi
Keyword(s):  
Tyrosine Phosphorylation ◽  
Kinase Activity ◽  
Tnf Alpha ◽  
Human Neutrophils ◽  
Gm Csf ◽  
Mitogen Activated Protein ◽  
Molecular Masses ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony

The present study was undertaken to determine the identities and characteristics of proteins with molecular masses between 40 and 44 kDa whose tyrosine phosphorylation increases in human neutrophils following stimulation of these cells with tumour necrosis factor alpha (TNF-alpha) and granulocyte-macrophage colony-stimulating factor (GM-CSF). Immunoblotting results demonstrate that addition of GM-CSF to human neutrophils increases the tyrosine phosphorylation of two proteins with molecular masses of 42 and 44 kDa. However, the addition of TNF-alpha to neutrophils induces a time- and dose-dependent increase in tyrosine phosphorylation of a 40 kDa protein. Immunoprecipitation using specific mitogen-activated protein kinase (MAPK) isoform antibodies and an antibody which recognizes phosphotyrosine-containing proteins demonstrated that the 42 and 44 kDa proteins are isoforms of MAPKs. Utilizing an in situ gel kinase activity assay, GM-CSF increases the kinase activity of the 42 and 44 kDa proteins. Moreover, using immunoprecipitated p42 and p44 MAPK isoforms in this gel assay revealed activity associated with the p42 and p44 MAPK isoforms. Using the same in situ assay, TNF-alpha induces an increase in kinase activity of a 40-42 kDa protein. However, the 40 kDa protein whose phosphorylation on tyrosine residues increased in human neutrophils following stimulation with TNF-alpha is not a member of the known MAPK family, demonstrating the divergences in pathways utilized by GM-CSF and TNF-alpha. This 40 kDa protein may be related to the recently identified protein that becomes phosphorylated on tyrosine residues upon stimulation of the human epidermal carcinoma cell line KB by interleukin-1. In these cells the p40 protein is part of a protein kinase cascade which results in the phosphorylation of the small heat shock protein, hsp27.

TNF-alpha and cyclooxygenase metabolites do not modulate C. albicans septic shock with disseminated candidiasis

1993 ◽  
Vol 74 (5) ◽  
pp. 2432-2442 ◽  
Author(s):  
G. M. Matuschak ◽  
C. A. Klein ◽  
T. L. Tredway ◽  
D. R. Schilly ◽  
A. J. Lechner
Keyword(s):  
Septic Shock ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Passive Immunization ◽  
Organ Injury ◽  
Tnf Alpha ◽  
Enzyme Linked Immunosorbent Assay ◽  
Necrosis Factor Alpha ◽  
Disseminated Candidiasis ◽  
Alpha Production

We analyzed differences in host regulation of tumor necrosis factor-alpha (TNF-alpha) production and pathophysiological responses in conscious rats after infection with two strains of pathogenic Candida albicans spp. (CA-1 and CA-2) compared with Escherichia coli serotype 055:B5 (EC). The hypothesis was tested that, in contrast to EC, hypotension, organ injury, and mortality after candidemia are not obligatorily dependent on TNF-alpha or TNF-alpha-induced cyclooxygenase pathway metabolites. Dose, viability, and strain-specific dependencies were established after intravenous 10(6) or 10(9) viable CA, as well as heat-killed (HK) or Formalin-inactivated (FI) CA blastospores, compared with live EC at the 24-h LD25 [10(9) colony-forming units (CFU)] and LD100 (10(10) CFU). Shock without endotoxemia developed 4–8 h after 10(9) live CA-1 or CA-2 (LD100 at 24 h) with disseminated yeast-mycelial transformation and increased microvascular permeability in multiple organs but not after HK or FI CA-1. Peak serum TNF-alpha after an LD100 of CA-1 or CA-2 was < 3% of LD25 EC values and was < 1% of peak values during lethal bacteremia. Similar pathogen-specific differences were found in liver- and lung-associated TNF. Production of functionally inactive TNF-alpha during candidemia was excluded by enzyme-linked immunosorbent assay and sodium dodecyl sulfate-polyacrylamide gel electrophoresis with Western blotting. Passive immunization against TNF-alpha 2 h before microbial challenge was not protective against CA but prevented otherwise lethal EC sepsis. Cyclooxygenase inhibition also failed to attenuate candidemic shock. We conclude that the magnitude and kinetics of TNF-alpha production and TNF-alpha-dependent immunophysiological responses are differentially regulated after lethal fungal vs. gram-negative bacterial infection. Thus TNF-alpha is not a pivotal mediator of the acute Candida septic shock syndrome with disseminated candidiasis.

scholarly journals Selective activation of p42 mitogen-activated protein (MAP) kinase in murine B lymphoma cell lines by membrane immunoglobulin cross-linking. Evidence for protein kinase C-independent and -dependent mechanisms of activation

1992 ◽  
Vol 287 (1) ◽  
pp. 269-276 ◽  
Author(s):  
M R Gold ◽  
J S Sanghera ◽  
J Stewart ◽  
S L Pelech
Keyword(s):  
Map Kinase ◽  
Tyrosine Phosphorylation ◽  
Cell Lines ◽  
Map Kinases ◽  
Phorbol Esters ◽  
Cross Linking ◽  
Kinase Activation ◽  
Membrane Immunoglobulin ◽  
Protein Map ◽  
Mitogen Activated Protein

Cross-linking of membrane immunoglobulin (mIg), the B lymphocyte antigen receptor, with anti-receptor antibodies stimulates tyrosine phosphorylation of a number of proteins, including one of 42 kDa. Proteins with a similar molecular mass are tyrosine-phosphorylated in response to receptor stimulation in other cell types and have been identified as serine/threonine kinases, termed mitogen-activated protein (MAP) kinases or extracellular signal-regulated kinases (ERKs). The MAP kinases constitute a family of related kinases, at least three of which have molecular masses of 40-45 kDa. In this paper we show that mIg cross-linking stimulated the myelin basic protein phosphotransferase activity characteristic of MAP kinase in both mature and immature murine B cell lines. This enzyme activity co-purified on three different columns with a 42 kDa protein that was tyrosine-phosphorylated (pp42) in response to mIg cross-linking and which reacted with a panel of anti-(MAP kinase) antibodies. Although immunoblotting with the anti-(MAP kinase) antibodies showed that these B cell lines expressed both 42 kDa and 44 kDa forms of MAP kinase, only the 42 kDa form was activated and tyrosine-phosphorylated to a significant extent. Activation of protein kinase C (PKC) with phorbol esters also resulted in selective tyrosine phosphorylation and activation of the 42 kDa MAP kinase. This suggested that mIg-induced MAP kinase activation could be due to stimulation of PKC by mIg. However, mIg-stimulated MAP kinase activation and pp42 tyrosine phosphorylation was only partially blocked by a PKC inhibitor, the staurosporine analogue Compound 3. In contrast, Compound 3 completely blocked the ability of phorbol esters to stimulate MAP kinase activity and induce tyrosine phosphorylation of pp42. Thus mIg may activate MAP kinase by both PKC-dependent and -independent mechanisms.

scholarly journals Lipopolysaccharide-induced selective priming effects on tumor necrosis factor alpha and nitric oxide production in mouse peritoneal macrophages.

1993 ◽  
Vol 177 (2) ◽  
pp. 511-516 ◽  
Author(s):  
X Zhang ◽  
D C Morrison
Keyword(s):  
Tumor Necrosis ◽  
Peritoneal Macrophages ◽  
Tnf Alpha ◽  
No Production ◽  
Necrosis Factor Alpha ◽  
Priming Effects ◽  
Alpha Production ◽  
Factor Alpha ◽  
Mouse Peritoneal Macrophages ◽  
Necrosis Factor

Preculture of thioglycollate-elicited C3HeB/FeJ mouse peritoneal macrophages in vitro with subthreshold stimulatory concentrations of lipopolysaccharide (LPS) can induce hyporesponsiveness (desensitization) to both tumor necrosis factor alpha (TNF-alpha) and nitric oxide (NO) production when these cells are subsequently stimulated with 100 ng/ml of LPS. We have established, however, that the primary dose of LPS required for inducing downregulation of NO production is significantly lower than that required for inducing downregulation of TNF-alpha production. Further, when LPS-pretreated macrophages become refractory to subsequent LPS stimulation for NO production, the secondary LPS-stimulated TNF-alpha production is markedly enhanced, and vice versa. These results indicate that LPS-induced TNF-alpha and NO production by macrophages are differentially regulated, and that the observed desensitization process may not reflect a state in which macrophages are totally refractory to subsequent LPS stimulation. Rather, our data suggest that LPS-pretreated macrophages become selectively primed for differential responses to LPS. The LPS-induced selective priming effects are not restricted to LPS stimulation, but extend as well to stimuli such as zymosan, Staphylococcus aureus, and heat-killed Listeria monocytogenes.

scholarly journals Polymorphisms in the tumor necrosis factor alpha (TNF-alpha) gene correlate with murine resistance to development of toxoplasmic encephalitis and with levels of TNF-alpha mRNA in infected brain tissue.

1992 ◽  
Vol 175 (3) ◽  
pp. 683-688 ◽  
Author(s):  
Y R Freund ◽  
G Sgarlato ◽  
C O Jacob ◽  
Y Suzuki ◽  
J S Remington
Keyword(s):  
Brain Tissue ◽  
Tumor Necrosis ◽  
Toxoplasmic Encephalitis ◽  
Tnf Alpha ◽  
Necrosis Factor Alpha ◽  
Alpha Production ◽  
D Region ◽  
Factor Alpha ◽  
Alpha Gene ◽  
Necrosis Factor

Murine resistance to development of toxoplasmic encephalitis (TE) has recently been mapped to the D region of the major histocompatibility complex (H-2). Since the gene for tumor necrosis factor alpha (TNF-alpha) is located 5' of the D region and TNF-alpha has been implicated as playing a role in neurological diseases, we were interested in determining the relationship of TNF-alpha production to TE resistance. We have demonstrated that resistance to TE in inbred mice can be correlated with specific restriction fragment length polymorphisms and microsatellite variants in the TNF-alpha gene. Mice that are susceptible to TE express elevated levels of TNF-alpha mRNA in brain tissue 6 wk after infection with the ME49 strain of Toxoplasma gondii. Resistant mice and all mice that are uninfected show no detectable TNF-alpha mRNA expression in brain tissue. Differences in the TNF-alpha gene between susceptible and resistant mice have been localized to the first intron, the promoter, and the 3' end of the TNF-alpha gene. These data implicate differences in regulation of TNF-alpha production in brain tissue as contributing to differences in susceptibility to development of TE.

scholarly journals Role of p38 in the Priming of Human Neutrophils by Peritoneal Dialysis Effluent

1999 ◽  
Vol 6 (6) ◽  
pp. 878-884 ◽  
Author(s):  
Ian Daniels ◽  
John Fletcher ◽  
Andrew Paul Haynes
Keyword(s):  
Arachidonic Acid ◽  
Peritoneal Dialysis ◽  
Map Kinases ◽  
Maximal Activity ◽  
Human Neutrophils ◽  
P38 Kinase ◽  
Necrosis Factor Alpha ◽  
Tnf Α ◽  
Mitogen Activated Protein ◽  
Peritoneal Dialysis Effluent

ABSTRACT Peritoneal dialysis effluent (PDE) contains a low-molecular-weight substance that is able to prime human neutrophils for the release of arachidonic acid and superoxide anion. Conventional priming agents, such as tumor necrosis factor alpha (TNF-α), are known to signal via mitogen-activated protein (MAP) kinases; at least one possible substrate for MAP kinases is cytosolic phospholipase A2(cPLA2). Phosphorylation of this enzyme results in arachidonic acid release, and this fatty acid is a potent primer and activator of the human neutrophil NADPH oxidase. Because of the striking similarities between the priming of neutrophils with agents such as TNF-α and PDE, we have investigated the signalling pathways evoked by PDE and explored the possibility that cPLA2 is a target for activated MAP kinases. Our results show that PDE treatment of human neutrophils results in the phosphorylation of the p38 kinase rather than the p42 and p44 kinases. Phosphorylation of p38 is transient with maximal activity being observed 1 min after exposure to PDE. We were unable to demonstrate that activation of p38 resulted in phosphorylation of cPLA2; furthermore, translocation of this enzyme to a membrane-containing fraction was not enhanced in PDE-treated neutrophils. Taken together, these data suggest that, in a manner similar to that of TNF-α, PDE primes human neutrophils by the activation of the p38 kinase. However, unlike the cytokine, the activation of this protein does not result in phosphorylation or activation of cPLA2.

scholarly journals YopJ of Yersinia spp. Is Sufficient To Cause Downregulation of Multiple Mitogen-Activated Protein Kinases in Eukaryotic Cells

1999 ◽  
Vol 67 (2) ◽  
pp. 708-716 ◽  
Author(s):  
Lance E. Palmer ◽  
Alessandra R. Pancetti ◽  
Steven Greenberg ◽  
James B. Bliska
Keyword(s):  
Map Kinase ◽  
Map Kinases ◽  
Cell Interaction ◽  
Small Gtpase ◽  
Effector Proteins ◽  
Eukaryotic Cells ◽  
Cell Periphery ◽  
Necrosis Factor Alpha ◽  
Type Iii ◽  
Mitogen Activated Protein

ABSTRACT Pathogenic Yersinia spp. utilize a plasmid-encoded type III secretion system to deliver a set of Yop effector proteins into eukaryotic cells. Previous studies have shown that the effector YopJ is required for Yersinia to cause downregulation of the mitogen-activated protein (MAP) kinases c-Jun N-terminal kinase (JNK), p38, and extracellular signal-regulated kinase (ERK) 1 and 2 in infected macrophages. Here we demonstrate that YopJ is sufficient to cause downregulation of multiple MAP kinases in eukaryotic cells. Cellular fractionation experiments confirmed that YopJ is delivered into the cytoplasmic fraction of macrophages by the type III system. Production of YopJ in COS-1 cells by transfection significantly reduced (5- to 10-fold) activation of JNK, p38, and ERK in response to several different stimuli, including serum and tumor necrosis factor alpha. JNK activation mediated by RacV12, an activated mutant of Rac1, was also blocked by YopJ in COS-1 cells, indicating that YopJ acts downstream of this small GTPase to downregulate MAP kinase signaling. Analysis of transfected COS-1 cells by immunofluorescence microscopy revealed that YopJ is recruited from the cytoplasmic compartment to the cell periphery in response to stimuli (e.g., serum) that induce membrane ruffling. These data indicate that YopJ functions as a “MAP kinase toxin” to selectively block nuclear responses that are triggered byYersinia-host cell interaction.

Protective effect of mepacrine on hypoxia-reoxygenation-induced acute lung injury in rats

1995 ◽  
Vol 78 (1) ◽  
pp. 225-231 ◽  
Author(s):  
C. Y. Shen ◽  
D. Wang ◽  
M. L. Chang ◽  
K. Hsu
Keyword(s):  
Acute Lung Injury ◽  
Lung Injury ◽  
Protective Effect ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Tnf Alpha ◽  
Lung Weight ◽  
Necrosis Factor Alpha ◽  
Alpha Production ◽  
Hypoxia Reoxygenation

Mepacrine, a cell membrane stabilizer and inhibitor of phospholipase A2 (PLA2), exerts a protective effect on ischemia-reperfusion injury in heart; however, its effect in lungs has not been examined. This study aimed to determine whether mepacrine pretreatment attenuates ischemia-reperfusion lung injury simulated by hypoxia reoxygenation and to identify possible mechanisms for such protection. Acute lung injury was induced in Sprague-Dawley rats by ventilation with 5% CO2–95% N2 and 5% CO2–95% air. Pretreatment with 0.06 mM mepacrine significantly attenuated the acute lung injury. Capillary filtration coefficient, lung weight gain, and protein concentration of lung lavage fluid were significantly lower in mepacrine-treated rats than in rats exposed to hypoxia reoxygenation alone. Steroid dexamethasone, another potential PLA2 inhibitor, had almost no protective effect. Mepacrine but not dexamethasone caused dose-dependent attenuation of the increase in leukocyte chemiluminescence produced by exposure to phorbol myristate acetate. Mepacrine also dose-dependently inhibited production of tumor necrosis factor-alpha (TNF-alpha) by human monocytes; dexamethasone was much less effective in decreasing TNF-alpha production. We conclude that mepacrine but not dexamethasone can significantly attenuate a hypoxia-reoxygenation-induced injury of the lung. This protective effect of mepacrine may not be the result of its inhibition of PLA2 but rather of its downregulation of oxygen radical production by circulating or resident leukocytes or its attenuation of TNF-alpha production by macrophages.

scholarly journals Macrophage priming and lipopolysaccharide-triggered release of tumor necrosis factor alpha during graft-versus-host disease.

1992 ◽  
Vol 175 (2) ◽  
pp. 405-413 ◽  
Author(s):  
F P Nestel ◽  
K S Price ◽  
T A Seemayer ◽  
W S Lapp
Keyword(s):  
Tumor Necrosis Factor ◽  
Tumor Necrosis Factor Alpha ◽  
Tumor Necrosis ◽  
Acute Gvhd ◽  
Tnf Alpha ◽  
Triggered Release ◽  
Necrosis Factor Alpha ◽  
Alpha Production ◽  
Factor Alpha ◽  
Necrosis Factor

In this report we have investigated macrophage (M phi) activity and tumor necrosis factor alpha (TNF-alpha) production during graft-vs.-host disease (GVHD). TNF-alpha production by M phi requires two signals: priming of M phi by interferon followed by triggering of TNF-alpha production and release by lipopolysaccharide (LPS). The state of M phi activation was examined in nonirradiated B6AF1 recipient mice injected with either 60 x 10(6) (acute GVHD) or 30 x 10(6) (nonlethal GVHD) parental B6 lymphoid cells. During the early phase of acute GVHD, administration of normally sublethal amounts of LPS-triggered release of significant amounts of TNF-alpha into the serum resulting in death of the animals within 36 h. Normal animals treated with the same dose of LPS neither died nor produced detectable amounts of serum TNF-alpha. In vitro studies demonstrated that M phi were primed during GVHD. The level of M phi priming was greater during acute GVHD than nonlethal GVHD since 100-fold less LPS was required to trigger killing of a TNF-alpha-sensitive cell line by M phi from acute GVHD animals. The amount of TNF-alpha released into the serum after LPS injection increased during the course of the GVHD and was significantly greater in acute GVH-reactive mice. Endogenous LPS was detected in the serum of acute GVH-reactive animals coincident with the onset of mortality. The data provide evidence that during GVHD M phi are primed as a result of the allogeneic reaction and that endogenous LPS therefore triggers M phi production of TNF-alpha resulting in the symptoms characteristic of acute GVHD.

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