Pronase destroys the lipopolysaccharide receptor CD14 on Kupffer cells

1999 ◽  
Vol 276 (3) ◽  
pp. G591-G598 ◽  
Author(s):  
Kenichi Ikejima ◽  
Nobuyuki Enomoto ◽  
Vitor Seabra ◽  
Ayako Ikejima ◽  
David A. Brenner ◽  
...  
Keyword(s):  
Intracellular Calcium ◽  
Kupffer Cells ◽  
Peritoneal Macrophages ◽  
Raw 264.7 Cells ◽  
Rat Serum ◽  
Tnf Α ◽  
Collagenase Perfusion ◽  
Factor Α ◽  
Serum Dependent ◽  
In Cells

CD14 is a lipopolysaccharide (LPS) receptor distributed largely in macrophages, monocytes, and neutrophils; however, the role of CD14 in activation of Kupffer cells by LPS remains controversial. The purpose of this study was to determine if different methods used to isolate Kupffer cells affect CD14. Kupffer cells were isolated by collagenase (0.025%) or collagenase-Pronase (0.02%) perfusion and differential centrifugation using Percoll gradients and cultured for 24 h before experiments. CD14 mRNA was detected by RT-PCR from Kupffer cell total RNA as well as from peritoneal macrophages. Western blotting showed that Kupffer cells prepared with collagenase possess CD14; however, it was absent in cells obtained by collagenase-Pronase perfusion. Intracellular calcium in Kupffer cells prepared with collagenase was increased transiently to levels around 300 nM by addition of LPS with 5% rat serum, which contains LPS binding protein. This increase in intracellular calcium was totally serum dependent. Moreover, LPS-induced increases in intracellular calcium in Kupffer cells were blunted significantly (40% of controls) when cells were treated with phosphatidylinositol-specific phospholipase C, which cleaves CD14 from the plasma membrane. However, intracellular calcium did not increase when LPS was added to cells prepared by collagenase-Pronase perfusion even in the presence of serum. These cells were viable, however, because ATP increased intracellular calcium to the same levels as cells prepared with collagenase perfusion. Tumor necrosis factor-α (TNF-α) mRNA was increased in Kupffer cells prepared with collagenase perfusion 1 h after addition of LPS, an effect potentiated over twofold by serum; however, serum did not increase TNF-α mRNA in cells isolated via collagenase-Pronase perfusion. Moreover, treatment with Pronase rapidly decreased CD14 on mouse macrophages (RAW 264.7 cells) and Kupffer cells. These findings indicate that Pronase cleaves CD14 from Kupffer cells, whereas collagenase perfusion does not, providing an explanation for why Kupffer cells do not exhibit a CD14-mediated pathway when prepared with procedures using Pronase. It is concluded that Kupffer cells indeed contain a functional CD14 LPS receptor when prepared gently.

scholarly journals LPS receptor CD14 participates in release of TNF-α in RAW 264.7 and peritoneal cells but not in Kupffer cells

1998 ◽  
Vol 275 (1) ◽  
pp. G39-G46 ◽  
Author(s):  
Steven N. Lichtman ◽  
Jian Wang ◽  
John J. Lemasters
Keyword(s):  
Kupffer Cells ◽  
The Body ◽  
Raw 264.7 Cells ◽  
Raw 264.7 ◽  
Fixed Tissue ◽  
Tissue Macrophage ◽  
Peritoneal Cells ◽  
Tnf Α ◽  
Surface Receptor ◽  
Factor Α

Lipopolysaccharide (LPS) is a bacterial polymer that stimulates macrophages to release tumor necrosis factor-α (TNF-α). In macrophages (RAW 264.7 and peritoneal cells), LPS binds to the CD14 surface receptor as the first step toward signaling. Liver macrophages, Kupffer cells, are the most numerous fixed-tissue macrophage in the body. The presence of CD14 on Kupffer cells and its role in LPS stimulation of TNF-α were examined. TNF-α release by Kupffer cells after LPS stimulation was the same in the presence and absence of serum. RAW 264.7 and peritoneal cells, which utilize the CD14 receptor, released significantly less TNF-α after LPS stimulation in the absence of serum because of the absence of LPS-binding protein. Phosphatidylinositol-phospholipase C treatment, which cleaves the CD14 receptor, decreased LPS-stimulated TNF-α release by RAW 264.7 cells but not by Kupffer cells. Deacylated LPS (dLPS) competes with LPS at the CD14 receptor when incubated in a ratio of 100:1 (dLPS/LPS). Such competition blocked LPS-stimulated TNF-α release from RAW 264.7 cells but not from Kupffer cells. Western and fluorescence-activated cell sorter analysis directly demonstrated the presence of CD14 on RAW 264.7 cells and murine peritoneal cells but showed only minimal amounts of CD14 in murine Kupffer cells. LPS stimulation did not increase the amount of CD14 detectable on mouse Kupffer cells. CD14 expression is very low in Kupffer cells, and LPS-stimulated TNF-α release is independent of CD14 in these cells.

Hypoxia enhances lysosomal TNF-α degradation in mouse peritoneal macrophages

2008 ◽  
Vol 295 (1) ◽  
pp. C2-C12 ◽  
Author(s):  
Nitza Lahat ◽  
Michal A. Rahat ◽  
Amalia Kinarty ◽  
Lea Weiss-Cerem ◽  
Sigalit Pinchevski ◽  
...  
Keyword(s):  
Peritoneal Macrophages ◽  
Raw 264.7 Cells ◽  
Raw 264.7 ◽  
Mrna Levels ◽  
Tnf Α ◽  
Primary Mouse ◽  
Factor Α ◽  
Secretory Lysosomes ◽  
Mouse Peritoneal Macrophages ◽  
Necrosis Factor

Infection, simulated by lipopolysaccharide (LPS), is a potent stimulator of tumor necrosis factor-α (TNF-α) production, and hypoxia often synergizes with LPS to induce higher levels of the secreted cytokine. However, we show that in primary mouse peritoneal macrophages and in three mouse peritoneal macrophage cell lines (RAW 264.7, J774A.1, and PMJ-2R), hypoxia (O2 < 0.3%) reduces the secretion of LPS-induced TNF-α ( P < 0.01). In RAW 264.7 cells this reduction was not regulated transcriptionally as TNF-α mRNA levels remained unchanged. Rather, hypoxia and LPS reduced the intracellular levels of TNF-α by twofold ( P < 0.01) by enhancing its degradation in the lysosomes and inhibiting its secretion via secretory lysosomes, as shown by confocal microscopy and verified by the use of the lysosome inhibitor Bafilomycin A1. In addition, although hypoxia did not change the accumulation of the soluble receptor TNF-RII, it increased its binding to the secreted TNF-α by twofold ( P < 0.05). We suggest that these two posttranslational regulatory checkpoints coexist in hypoxia and may partially explain the reduced secretion and diminished biological activity of TNF-α in hypoxic peritoneal macrophages.

Production of superoxide and TNF-α from alveolar macrophages is blunted by glycine

1999 ◽  
Vol 277 (5) ◽  
pp. L952-L959 ◽  
Author(s):  
Michael D. Wheeler ◽  
Ronald G. Thurman
Keyword(s):  
Intracellular Calcium ◽  
Kupffer Cells ◽  
Alveolar Macrophages ◽  
Chloride Channel ◽  
Chloride Channels ◽  
Dependent Manner ◽  
Tnf Α ◽  
The Central Nervous System ◽  
Factor Α ◽  
Dose Dependent

Glycine blunts lipopolysaccharide (LPS)-induced increases in intracellular calcium concentration ([Ca2+]i) and tumor necrosis factor-α (TNF-α) production by Kupffer cells through a glycine-gated chloride channel. Alveolar macrophages, which have a similar origin as Kupffer cells, play a significant role in the pathogenesis of several lung diseases including asthma, endotoxemia, and acute inflammation due to inhaled bacterial particles and dusts. Therefore, studies were designed here to test the hypothesis that alveolar macrophages could be inactivated by glycine via a glycine-gated chloride channel. The ability of glycine to prevent endotoxin [lipopolysaccharide (LPS)]-induced increases in [Ca2+]iand subsequent production of superoxide and TNF-α in alveolar macrophages was examined. LPS caused a transient increase in intracellular calcium to nearly 200 nM, with EC50values slightly greater than 25 ng/ml. Glycine, in a dose-dependent manner, blunted the increase in [Ca2+]i, with an IC50less than 100 μM. Like the glycine-gated chloride channel in the central nervous system, the effects of glycine on [Ca2+]iwere both strychnine sensitive and chloride dependent. Glycine also caused a dose-dependent influx of radiolabeled chloride with EC50values near 10 μM, a phenomenon which was also inhibited by strychnine (1 μM). LPS-induced superoxide production was also blunted in a dose-dependent manner by glycine and was reduced ∼50% with 10 μM glycine. Moreover, TNF-α production was also inhibited by glycine and also required nearly 10 μM glycine for half-inhibition. These data provide strong pharmacological evidence that alveolar macrophages contain glycine-gated chloride channels and that their activation is protective against the LPS-induced increase in [Ca2+]iand subsequent production of toxic radicals and cytokines.

scholarly journals Production of cytokines by cell-effectors of natural cytotoxicity system in mice under development of Lewis lung carcinoma

2004 ◽  
Vol 3 (2) ◽  
pp. 24-29
Author(s):  
Ye. Yu. Sherstoboyev ◽  
O. A. Kaplya ◽  
Ye. P. Zuyeva ◽  
T. G. Razina ◽  
O. I. Epstein
Keyword(s):  
Lung Carcinoma ◽  
Lewis Lung Carcinoma ◽  
Tumor Development ◽  
Peritoneal Macrophages ◽  
Interferon Γ ◽  
Lewis Lung ◽  
Natural Cytotoxicity ◽  
Tnf Α ◽  
Factor Α ◽  
T Helpers

Cytokine production by cell-effectors of natural cytotoxicity system under Lewis lung carcinoma development in F1(CBAxC57B1/6) line mice has been studied. It has been revealed the increase of interleukin-1β (IL-1β) production and tumor necrosis factor-α (TNF-α) by peritoneal macrophages. At this the balance of cytokines produced by T-helpers (Th)  has  been displaced to Th2 side, IL-4 production has increased and interferon-γ (IFN-γ) and IL-2 production has decreased. The rise of IL-10 production by lymphocytes has been observed in the later terms of tumor development.

scholarly journals Lipopolysaccharide- TLR-4 Axis regulates Osteoclastogenesis independent of RANKL/RANK signaling

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Mohammed S. AlQranei ◽  
Linda T. Senbanjo ◽  
Hanan Aljohani ◽  
Therwa Hamza ◽  
Meenakshi A. Chellaiah
Keyword(s):  
Tumor Necrosis Factor ◽  
Tumor Necrosis ◽  
Osteoclast Differentiation ◽  
Bone Diseases ◽  
Raw 264.7 Cells ◽  
Gram Negative ◽  
Tnf Α ◽  
Necrosis Factor ◽  
In Cells

Abstract Background Lipopolysaccharide (LPS) is an endotoxin and a vital component of gram-negative bacteria’s outer membrane. During gram-negative bacterial sepsis, LPS regulates osteoclast differentiation and activity, in addition to increasing inflammation. This study aimed to investigate how LPS regulates osteoclast differentiation of RAW 264.7 cells in vitro. Results Herein, we revealed that RAW cells failed to differentiate into mature osteoclasts in vitro in the presence of LPS. However, differentiation occurred in cells primed with receptor activator of nuclear factor-kappa-Β ligand (RANKL) for 24 h and then treated with LPS for 48 h (henceforth, denoted as LPS-treated cells). In cells treated with either RANKL or LPS, an increase in membrane levels of toll-like receptor 4 (TLR4) receptor was observed. Mechanistically, an inhibitor of TLR4 (TAK-242) reduced the number of osteoclasts as well as the secretion of tumor necrosis factor (TNF)-α in LPS-treated cells. RANKL-induced RAW cells secreted a very basal level TNF-α. TAK-242 did not affect RANKL-induced osteoclastogenesis. Increased osteoclast differentiation in LPS-treated osteoclasts was not associated with the RANKL/RANK/OPG axis but connected with the LPS/TLR4/TNF-α tumor necrosis factor receptor (TNFR)-2 axis. We postulate that this is because TAK-242 and a TNF-α antibody suppress osteoclast differentiation. Furthermore, an antibody against TNF-α reduced membrane levels of TNFR-2. Secreted TNF-α appears to function as an autocrine/ paracrine factor in the induction of osteoclastogenesis independent of RANKL. Conclusion TNF-α secreted via LPS/TLR4 signaling regulates osteoclastogenesis in macrophages primed with RANKL and then treated with LPS. Our findings suggest that TLR4/TNF-α might be a potential target to suppress bone loss associated with inflammatory bone diseases, including periodontitis, rheumatoid arthritis, and osteoporosis.

scholarly journals Thiol regulation of endotoxin-induced release of tumour necrosis factor α from isolated rat Kupffer cells

1996 ◽  
Vol 320 (3) ◽  
pp. 1005-1010 ◽  
Author(s):  
Brent A. NEUSCHWANDER-TETRI ◽  
Joseph M. BELLEZZO ◽  
Robert S. BRITTON ◽  
Bruce R. BACON ◽  
Eben S. FOX
Keyword(s):  
Tumour Necrosis Factor ◽  
Kupffer Cells ◽  
Tumour Necrosis ◽  
Oxidant Stress ◽  
Antioxidant Properties ◽  
Tumour Necrosis Factor Α ◽  
General Role ◽  
Tnf Α ◽  
Factor Α ◽  
Necrosis Factor

Proinflammatory cytokines released by hepatic macrophages (Kupffer cells) have a central role in the pathogenesis of liver injury and the cardiovascular abnormalities of sepsis. Because cytokine release is controlled primarily at the level of gene expression, intracellular signalling mechanisms that control the transcription of cytokine genes are critical links to organ injury. Oxidant stress up-regulates and antioxidants down-regulate the pleiotropic transcription factor NF-κB, a DNA-binding protein that induces the expression of cytokines and vascular adhesion molecules. Thiol-bearing molecules are also important inhibitors of NF-κB activation, but whether this inhibition represents an antioxidant effect is unknown. This study was undertaken to determine whether important endogenous and pharmacological thiols modulate the activation of NF-κB and the release of tumour necrosis factor α (TNF-α) from Kupffer cells and to ascertain whether these effects are mediated through glutathione. Exposure of rat Kupffer cells to a physiologically relevant concentration of lipopolysaccharide (10 ng/ml) activated NF-κB within 1 h and induced the release of TNF-α over 5 h. Cellular glutathione content remained unchanged after lipopolysaccharide exposure, but both glutathione monoethyl ester and N-acetyl-l-cysteine increased cellular glutathione levels, blocked NF-κB activation and inhibited the release of TNF-α. Inhibition of glutathione synthesis prevented the NAC-induced increase in Kupffer cell glutathione, yet it did not prevent the inhibition of TNF-α release by NAC. Thus the inhibition of NF-κB activation by pharmacological thiols such as NAC might reflect a more general role of the intracellular thiol redox status in NF-κB regulation rather than the antioxidant properties of these agents.

scholarly journals Increments in cytokines and matrix metalloproteinases in skeletal muscle after injection of tissue-damaging toxins from the venom of the snakeBothrops asper

2002 ◽  
Vol 11 (2) ◽  
pp. 121-128 ◽  
Author(s):  
Alexandra Rucavado ◽  
Teresa Escalante ◽  
Catarina F. P. Teixeira ◽  
Cristina María Fernándes ◽  
Cecilia Díaz ◽  
...  
Keyword(s):  
Matrix Metalloproteinases ◽  
Inflammatory Response ◽  
Experimental Model ◽  
Peritoneal Macrophages ◽  
Interferon Γ ◽  
Local Inflammatory Response ◽  
Mmp Inhibitor ◽  
Tnf Α ◽  
Factor Α

Envenomations by the snakeBothrops asperare characterized by prominent local tissue damage (i.e. myonecrosis), blistering, hemorrhage and edema. Various phospholipases A2and metalloproteinases that induce local pathological alterations have been purified from this venom. Since these toxins induce a conspicuous inflammatory response, it has been hypothesized that inflammatory mediators may contribute to the local pathological alterations described. This study evaluated the local production of cytokines and matrix metalloproteinases (MMPs) as a consequence of intramuscular injections of an Asp-49 myotoxic phospholipase A2(myotoxin III (MT-III)) and a P-I type hemorrhagic metalloproteinase (BaP1) isolated fromB. aspervenom. Both enzymes induced prominent tissue alterations and conspicuous increments in interleukin (IL)-1β, IL-6 and a number of MMPs, especially gelatinase MMP-9, rapidly after injection. In contrast, no increments in tumor necrosis factor-α (TNF-α) and interferon-γ were detected. In agreement, MT-III and BaP1 did not induce the synthesis of TNF-α by resident peritoneal macrophagesin vitro. Despite the conspicuous expression of latent forms of MMPs in muscle, evidenced by zymography, there were no increments in activated MMP-2 and only a small increase in activated MMP-9, as detected by a functional enzymatic assay. This suggests that MMP activity was regulated by a highly controlled activation of latent forms and, probably, by a concomitant synthesis of MMP inhibitors. Since no hemorrhage nor dermonecrosis were observed after injection of MT-III, despite a prominent increase in MMP expression, and since inflammatory exudate did not enhance hemorrhage induced by BaP1, it is suggested that endogenous MMPs released in the tissue are not responsible for the dermonecrosis and hemorrhage characteristic ofB. asperenvenomation. Moreover, pretreatment of mice with the peptidomimetic MMP inhibitor batimastat did not reduce myotoxic nor edema-forming activities of MT-III, suggesting that MMPs do not play a prominent role in the pathogenesis of these effects in this experimental model. It is concluded that MT-III and BaP1 induce a local inflammatory response associated with the synthesis of IL-1β, IL-6 and MMPs. MMPs do not seem to play a prominent role in the acute local pathological alterations induced by these toxins in this experimental model.

scholarly journals C16, a novel sinomenine derivatives, promoted macrophage reprogramming toward M2-like phenotype and protected mice from endotoxemia

2021 ◽  
Vol 35 ◽  
pp. 205873842110267
Author(s):  
Ping Ni ◽  
Yue-Qin Liu ◽  
Jin-Yu Man ◽  
Wang Li ◽  
Shan-Shan Xue ◽  
...  
Keyword(s):  
Peritoneal Macrophages ◽  
Enzyme Linked Immunosorbent Assay ◽  
Phenotype Correlation ◽  
Arginase 1 ◽  
Tnf Α ◽  
Anti Inflammatory ◽  
Factor Α ◽  
Correlation Factors ◽  
M2 Phenotype

Macrophage plays a critical part in host defense, tissue repair, and anti-inflammation; Macrophage reprogramming is responsible for disease development or regression. We aimed to clarify the effect of sinomenine-4-hydroxy-palmitate (C16), on macrophage reprogramming and anti-inflammatory in endotoxemia model. According to a structure modification of SIN (Sinomenine), C16 was found. Then, based on the endotoxin model, the mice liver and kidney toxicity was evaluated and serum cytokines level of IL-6 (Interleukin-6), TNF-α (Tumor necrosis factor-α), and IL-1β (Interleukin-1β) were measured by ELISA (Enzyme linked immunosorbent assay). Then, we confirmed the effect of C16 on macrophages reprogramming, we used the flow cytometry to test the effect of C16 on macrophages apoptosis in vitro. Then, iNOS (Inducible nitric oxide synthase), M1-type related cytokines, such as IL-1β, TNF-α, and M2-type related cytokines, such as Arg-1 (Arginase-1), CD206, Fizz1, and Ym1 was detected, which expressed in ANA-1 and primary peritoneal macrophages. To further explore the molecular mechanism of C16 in reprogramming of macrophages from M1 toward M2 phenotype, the expression of STAT1 (signal transducer and activator of Transcription 1), STAT3, ERK1/2 (extracellular signal regulated kinase1/2), AKT, p38, and its corresponding phosphorylation were determined by western blot. Our results demonstrated that C16 improved the survival rate of LPS- (lipopolysaccharide) challenged mice and decreased the inflammatory cytokines expression; After C16 treatment, the expression of M1 phenotype correlation factors decreased significantly, while the expression of M2 phenotype correlation factors increased significantly at different levels compared with normal group. It indicated that C16 reprogram macrophages phenotype from M1 toward M2 following LPS stimulus. Furthermore, the results also showed that C16 showed anti-inflammatory effect by inhibiting LPS-induced p38, AKT and STAT1 phosphorylation and contributing ERK1/2 activation. C16 promoted macrophage reprogramming toward M2-like phenotype via p-p38/p-AKT or STAT1 signals pathway and C16 might be a valid candidate for inflammatory disease.

scholarly journals Taurine blunts LPS-induced increases in intracellular calcium and TNF-α production by Kupffer cells

1998 ◽  
Vol 64 (5) ◽  
pp. 615-621 ◽  
Author(s):  
Vítor Seabra ◽  
Robert F. Stachlewitz ◽  
Ronald G. Thurman
Keyword(s):  
Intracellular Calcium ◽  
Kupffer Cells ◽  
Tnf Α

Leonurus sibiricus induces nitric oxide and tumor necrosis factor-α in mouse peritoneal macrophages

2008 ◽  
Vol 86 (10) ◽  
pp. 682-690 ◽  
Author(s):  
Hyo-Jin An ◽  
Hong-Kun Rim ◽  
Jong-Hyun Lee ◽  
Se-Eun Suh ◽  
Ji-Hyun Lee ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Host Defense ◽  
Tumor Necrosis ◽  
Peritoneal Macrophages ◽  
No Production ◽  
Tnf Α ◽  
Leonurus Sibiricus ◽  
Factor Α ◽  
Mouse Peritoneal Macrophages ◽  
Necrosis Factor

Using mouse peritoneal macrophages, we have examined the mechanism by which Leonurus sibiricus (LS) regulates nitric oxide (NO) production. When LS was used in combination with recombinant interferon-γ (rIFN-γ), there was a marked cooperative induction of NO production; however, LS by itself had no effect on NO production. The increased production of NO from rIFN-γ plus LS-stimulated cells was almost completely inhibited by pretreatment with pyrrolidine dithiocarbamate (PDTC), an inhibitor of nuclear factor κB. Furthermore, treatment of peritoneal macrophages with rIFN-γ plus LS caused a significant increase in tumor necrosis factor-α (TNF-α) production. PDTC also decreased the effect of LS on TNF-α production significantly. Because NO and TNF-α play an important role in immune function and host defense, LS treatment could modulate several aspects of host defense mechanisms as a result of stimulation of the inducible nitric oxide synthase.

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