scholarly journals Differentiation of Monocytes to Macrophages Primes Cells for Lipopolysaccharide Stimulation via Accumulation of Cytoplasmic Nuclear Factor κB

1999 ◽  
Vol 67 (11) ◽  
pp. 5573-5578 ◽  
Author(s):  
Shogo Takashiba ◽  
Thomas E. Van Dyke ◽  
Salomon Amar ◽  
Yoji Murayama ◽  
Aubrey W. Soskolne ◽  
...  
Keyword(s):  
Nuclear Factor Κb ◽  
Nuclear Factor ◽  
Blood Monocytes ◽  
Monocytic Cell ◽  
Monocytic Cell Line ◽  
Necrosis Factor Alpha ◽  
Lps Challenge ◽  
Differentiated Cells ◽  
Human Monocytic Cell Line ◽  
Tnf Α

ABSTRACT During infection, circulating blood monocytes migrate from the vasculature to the extravascular compartments where they mature into tissue macrophages. The maturation process prepares the cell to actively participate in the inflammatory and the immune responses, and many transcription factors have been found to be involved. Here we report on a novel role for nuclear factor κB (NF-κB) in this process. Its accumulation in the cytoplasm of differentiated macrophages is responsible for the enhanced ability of the cell to respond to lipopolysaccharide (LPS) stimulation, as determined by tumor necrosis factor alpha (TNF-α) secretion. Differentiation of the human monocytic cell line THP-1 into macrophage-like cells was induced by exposure of the cells to phorbol myristate acetate. DNA-bindable NF-κB was not detected in the cytoplasm of undifferentiated THP-1 cells but accumulated in the cytoplasm of the cells following differentiation. No TNF-α was detected in the media of resting differentiated and nondifferentiated THP-1 cells. Stimulation with LPS of differentiated cells induced the production of higher levels of TNF-α than stimulation of nondifferentiated cells. This hyperresponsiveness to LPS was found in the mRNA and secreted TNF-α levels. Furthermore, stimulation with LPS induced the translocation of NF-κB from the cytoplasm into the nucleus. This translocation process was more rapid in the differentiated cells than in the nondifferentiated cells, and the resultant accumulated levels of NF-κB in the nucleus were higher. The DNA-bindable NF-κB was identified as a heterodimer of p65 and p50. The results suggest that NF-κB accumulation in the cytoplasm during maturation of monocytes to macrophages primes the cells for enhanced responsiveness to LPS and results in the rapid secretion of inflammatory mediators, such as TNF-α, by mature macrophages following LPS challenge.

scholarly journals Effects of Cytokines and Endotoxin on the Intracellular Growth of Bacteria

1999 ◽  
Vol 67 (6) ◽  
pp. 2834-2840 ◽  
Author(s):  
Siva Kanangat ◽  
G. Umberto Meduri ◽  
Elizabeth A. Tolley ◽  
David R. Patterson ◽  
Christopher U. Meduri ◽  
...  
Keyword(s):  
Proinflammatory Cytokines ◽  
Bacterial Infections ◽  
Human Monocytes ◽  
Blood Monocytes ◽  
Intracellular Growth ◽  
Phagocytic Cells ◽  
Monocytic Cell ◽  
Monocytic Cell Line ◽  
Necrosis Factor Alpha ◽  
Human Monocytic Cell Line

ABSTRACT Patients with unresolving acute respiratory distress syndrome (ARDS) have persistently elevated levels of proinflammatory cytokines in the lungs and circulation and increased rates of bacterial infections. Phagocytic cells hyperactivated with lipopolysaccharide (LPS), which induces high levels of proinflammatory cytokines in monocytic cells, are inefficient in killing ingested bacteria despite having intact phagocytic activity. On the other hand, phagocytic cells that are activated with an analogue of LPS that does not induce the expression of proinflammatory cytokines effectively ingest and kill bacteria. We hypothesized that in the presence of high concentrations of proinflammatory cytokines, bacteria may adapt and utilize cytokines to their growth advantage. To test our hypothesis, we primed a human monocytic cell line (U937) with escalating concentrations of the proinflammatory cytokines tumor necrosis factor alpha, interleukin-1β (IL-1β), and IL-6 and with LPS. These cells were then exposed to fresh isolates of three common nosocomial pathogens:Staphylococcus aureus, Pseudomonas aeruginosa, and an Acinetobacter sp. In human monocytes primed with lower concentrations of proinflammatory cytokines (10 to 250 pg) or LPS (1 and 10 ng), intracellular bacterial growth decreased. However, when human monocytes were primed with higher concentrations of proinflammatory cytokines (1 to 10 ng) or LPS (1 to 10 μg), intracellular growth of the tested bacteria increased significantly (P <0.0001). These results were reproduced with peripheral blood monocytes obtained from normal healthy volunteers. The specificity of the cytokine activity was demonstrated by neutralizing the cytokines with specific antibodies. Our findings provide a possible mechanism to explain the frequent development of bacterial infections in patients with an intense and protracted inflammatory response.

scholarly journals Haemophilus influenzae Porin Induces Toll-Like Receptor 2-Mediated Cytokine Production in Human Monocytes and Mouse Macrophages

2004 ◽  
Vol 72 (2) ◽  
pp. 1204-1209 ◽  
Author(s):  
Marilena Galdiero ◽  
Massimiliano Galdiero ◽  
Emiliana Finamore ◽  
Fabio Rossano ◽  
Maria Gambuzza ◽  
...  
Keyword(s):  
Haemophilus Influenzae ◽  
Cytokine Production ◽  
Inflammatory Responses ◽  
Adaptor Protein ◽  
Monocytic Cell ◽  
Monocytic Cell Line ◽  
Necrosis Factor Alpha ◽  
Human Monocytic Cell Line ◽  
Tnf Α ◽  
Human Monocytic Cell

ABSTRACT The production of proinflammatory cytokines is likely to play a major pathophysiological role in meningitis and other infections caused by Haemophilus influenzae type b (Hib). Previous studies have shown that Hib porin contributes to signaling of the inflammatory cascade. We examined here the role of Toll-like receptors (TLRs) and the TLR-associated adaptor protein MyD88 in Hib porin-induced production of tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6). Hib porin-induced TNF-α and IL-6 production was virtually eliminated in macrophages from TLR2- or MyD88-deficient mice. In contrast, macrophages from lipopolysaccharide (LPS)-hyporesponsive C3H/HeJ mice, which are defective in TLR4 function, responded normally to Hib porin. Moreover anti-TLR2 antibodies but not anti-TLR4 antibodies significantly reduced Hib porin-stimulated TNF-α and IL-6 release from the human monocytic cell line THP-1. These data indicate that the TLR2/MyD88 pathway plays an essential role in Hib porin-mediated cytokine production. These findings may be useful in the development of alternative therapies aimed at reducing excessive inflammatory responses during Hib infections.

scholarly journals Autocrine and Exocrine Regulation of Interleukin-10 Production in THP-1 Cells Stimulated with Borrelia burgdorferi Lipoproteins

2002 ◽  
Vol 70 (4) ◽  
pp. 1881-1888 ◽  
Author(s):  
Guillermo H. Giambartolomei ◽  
Vida A. Dennis ◽  
Barbara L. Lasater ◽  
P. K. Murthy ◽  
Mario T. Philipp
Keyword(s):  
Borrelia Burgdorferi ◽  
Interleukin 10 ◽  
Feedback Mechanism ◽  
Monocytic Cell ◽  
Monocytic Cell Line ◽  
Necrosis Factor Alpha ◽  
Negative Feedback Mechanism ◽  
Human Monocytic Cell Line ◽  
Tnf Α ◽  
Ifn Γ

ABSTRACT We have recently demonstrated that interleukin-10 (IL-10), produced by THP-1 monocytes in response to Borrelia burgdorferi lipoproteins, dampens the production of concomitantly elicited inflammatory cytokines. Thus, IL-10 could potentially down-regulate inflammatory and microbicidal effector mechanisms of the innate immune response to a B. burgdorferi infection, facilitating the establishment of the spirochete. To understand the mechanism(s) implicated in the regulation of the synthesis and release of IL-10 during early infection, we investigated the autocrine effects of IL-6, IL-12, tumor necrosis factor alpha (TNF-α), and IL-10 itself, as well as the exocrine effect of IFN-γ on the production of macrophage-derived IL-10 with lipoprotein as a stimulant. In addition, in view of the differences in the receptor and signal transduction pathways of lipopolysaccharide (LPS) and bacterial lipoproteins, we also investigated the effects described above with LPS as a stimulant. The THP-1 human monocytic cell line and purified recombinant lipidated OspA (L-OspA) were used as the model target cell and stimulant, respectively. TNF-α increased the production of IL-10, as elicited by lipoproteins. The production of IL-10 by THP-1 cells stimulated with L-OspA was autoregulated by a negative feedback mechanism involving the IL-10 receptor (IL-10R). Exogenous IFN-γ significantly inhibited the production of IL-10. Both autocrine (IL-10) and exocrine (IFN-γ) inhibition of IL-10 production resulted in an increase in the production of the proinflammatory cytokines IL-6 and IL-12. The same results were obtained when the stimulant was LPS. The results further illustrate that IL-10 may play a pivotal role in Lyme disease pathogenesis. Moreover, the regulation of its production with lipoprotein as a stimulant is indistinguishable from that observed when LPS acts as a stimulant.

scholarly journals Engagement of the Lewis X Antigen (CD15) Results in Monocyte Activation

Blood ◽  
1997 ◽  
Vol 89 (1) ◽  
pp. 307-314 ◽  
Author(s):  
Siu K. Lo ◽  
Douglas T. Golenbock ◽  
Philip M. Sass ◽  
Azmat Maskati ◽  
Hong Xu ◽  
...  
Keyword(s):  
Nuclear Factor Κb ◽  
Specific Protein ◽  
Cross Linking ◽  
Blood Monocytes ◽  
Monocyte Activation ◽  
Lewis X ◽  
Sv40 Promoter ◽  
Monocytic Cell ◽  
Monocytic Cell Line ◽  
Tnf Α

Abstract We previously reported that monocyte adhesion to tumor necrosis factor-α (TNF-α)–treated endothelial cells increased expression of tissue factor and CD36 on monocytes. Using immunological cross-linking to mimic receptor engagement by natural ligands, we now show that CD15 (Lewis X), a monocyte counter-receptor for endothelial selectins may participate in this response. We used cytokine production as a readout for monocyte activation and found that CD15 cross-linking induced TNF-α release from peripheral blood monocytes and cells from the monocytic cell line MM6. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) showed an increase in steady-state TNF-α mRNA after 3 to 4 hours of cross-linking. CD15 cross-linking also concomitantly increased interleukin-1β (IL-1β) mRNA, while no apparent change was observed in the levels of β-actin mRNA, indicating specificity. To examine transcriptional regulation of cytokine genes by CD15 engagement, a CAT plasmid reporter construct containing IL-1β promoter/enhancer sequences was introduced into MM6. Subsequent cross-linking of CD15 increased CAT activity. CD15 engagement by monoclonal antibody also attenuated IL-1β transcript degradation, demonstrating that signaling via CD15 also had posttranscriptional effects. Nuclear extracts of anti-CD15 cross-linked cells demonstrated enhanced levels of the transcriptional factor activator protein-1, minimally changed nuclear factor-κB, and did not affect SV40 promoter specific protein-1. We conclude that engagement of CD15 on monocytes results in monocyte activation. In addition to its well-recognized adhesive role, CD15 may function as an important signaling molecule capable of initiating proinflammatory events in monocytes that come into contact with activated endothelium.

scholarly journals Engagement of the Lewis X Antigen (CD15) Results in Monocyte Activation

Blood ◽  
1997 ◽  
Vol 89 (1) ◽  
pp. 307-314
Author(s):  
Siu K. Lo ◽  
Douglas T. Golenbock ◽  
Philip M. Sass ◽  
Azmat Maskati ◽  
Hong Xu ◽  
...  
Keyword(s):  
Nuclear Factor Κb ◽  
Specific Protein ◽  
Cross Linking ◽  
Blood Monocytes ◽  
Monocyte Activation ◽  
Lewis X ◽  
Sv40 Promoter ◽  
Monocytic Cell ◽  
Monocytic Cell Line ◽  
Tnf Α

We previously reported that monocyte adhesion to tumor necrosis factor-α (TNF-α)–treated endothelial cells increased expression of tissue factor and CD36 on monocytes. Using immunological cross-linking to mimic receptor engagement by natural ligands, we now show that CD15 (Lewis X), a monocyte counter-receptor for endothelial selectins may participate in this response. We used cytokine production as a readout for monocyte activation and found that CD15 cross-linking induced TNF-α release from peripheral blood monocytes and cells from the monocytic cell line MM6. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) showed an increase in steady-state TNF-α mRNA after 3 to 4 hours of cross-linking. CD15 cross-linking also concomitantly increased interleukin-1β (IL-1β) mRNA, while no apparent change was observed in the levels of β-actin mRNA, indicating specificity. To examine transcriptional regulation of cytokine genes by CD15 engagement, a CAT plasmid reporter construct containing IL-1β promoter/enhancer sequences was introduced into MM6. Subsequent cross-linking of CD15 increased CAT activity. CD15 engagement by monoclonal antibody also attenuated IL-1β transcript degradation, demonstrating that signaling via CD15 also had posttranscriptional effects. Nuclear extracts of anti-CD15 cross-linked cells demonstrated enhanced levels of the transcriptional factor activator protein-1, minimally changed nuclear factor-κB, and did not affect SV40 promoter specific protein-1. We conclude that engagement of CD15 on monocytes results in monocyte activation. In addition to its well-recognized adhesive role, CD15 may function as an important signaling molecule capable of initiating proinflammatory events in monocytes that come into contact with activated endothelium.

scholarly journals Palmitate-Induced MMP-9 Expression in the Human Monocytic Cells is Mediated through the TLR4-MyD88 Dependent Mechanism

2016 ◽  
Vol 39 (3) ◽  
pp. 889-900 ◽  
Author(s):  
Sardar Sindhu ◽  
Areej Al-Roub ◽  
Merin Koshy ◽  
Reeby Thomas ◽  
Rasheed Ahmad
Keyword(s):  
Underlying Mechanism ◽  
Positive Control ◽  
Monocytic Cell ◽  
Monocytic Cell Line ◽  
Monocytic Cells ◽  
Metabolic Inflammation ◽  
Human Monocytic Cell Line ◽  
Tnf Α ◽  
Human Monocytic Cell

Background/Aims: Obese individuals are known to have increased Matrix metalloproteinase (MMP)-9 plasma levels and MMP-9 is reported to play an important role in obesity-associated adipose tissue inflammation. Since in obesity, the levels of circulatory saturated free fatty acid (FFA) palmitate (palimitic acid) are increased and modulate the expression of inflammatory mediators, the role of palmitate in the regulation of MMP-9 remains unclear. Methods: Human monocytic cell line THP-1 and primary monocytes were stimulated with palmitate and TNF-α (positive control). MMP-9 expression was assessed with real time RT-PCR and ELISA. Signaling pathways were studied by using THP-1-XBlue™ cells, THP-1-XBlue™-defMyD cells, anti-TLR4 mAb and TLR4 siRNA. Phosphorylation of NF-kB and c-Jun was analyzed by Western blotting. Results: Here, we provide the evidence that palmitate induces MMP-9 expression at both mRNA (THP-1: 6.8 ± 1.2 Fold; P = 0.01; Primary monocytes: 5.9 ± 0.7 Fold; P = 0.0003) and protein (THP1: 1116 ±14 pg/ml; P<0.001; Primary monocytes: 1426 ± 13.8; P = 0.0005) levels in human monocytic cells. Palmitate-induced MMP-9 secretion was markedly suppressed by neutralizing anti-TLR-4 antibody (P < 0.05). Furthermore, genetic silencing of TLR4 by siRNA also significantly abrogated the palmitate-induced up-regulation of MMP-9. Additionally, MyD88-/- THP-1 cells did not express MMP-9 in response to palmitate treatment. Increased NF-κB/AP-1 activity (P<0.05) was also observed in palmitate-treated THP-1 cells. Conclusion: Altogether, these results show that palmitate induces TLR4-dependent activation of MMP-9 gene expression, which requires the recruitment of MyD88 leading to activation of NF-kB/AP-1 transcription factors. Thus, our findings suggest that the palmitate-induced MMP-9 secretion might be an underlying mechanism of its increased levels in obesity and related metabolic inflammation.

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