Janet Bellingham

Mechanism of decreased in vitro murine macrophage cytokine release after exposure to carbon dioxide: relevance to laparoscopic surgery.

OBJECTIVE The objective of this study was to determine the effect of carbon dioxide (CO2) on the function of peritoneal macrophages. SUMMARY BACKGROUND DATA Laparoscopic surgery is associated with minimal pain, fever, and low levels of inflammatory cytokines. To understand the mechanisms involved, the authors investigated the effect of different gases on murine peritoneal macrophage intracellular pH and correlated these alterations with alterations in LPS-stimulated inflammatory cytokine release. METHODS Peritoneal macrophages were incubated for 2 hours in air, helium, or CO2, and the effect of the test gas on immediate or next day lipopolysaccharide (LPS)-stimulated tumor necrosis factor (TNF) and interleukin-1 release compared. Cytosolic pH of macrophages exposed to test gases was measured using single-cell fluorescent imaging. The in vivo effects of test gases were determined in anesthetized rats during abdominal insufflation. RESULTS Macrophages incubated in CO2 produced significantly less TNF and interleukin-1 in response to LPS compared to incubation in air or helium. Cytokine production returned to normal 24 hours later. Exposure to CO2, but not air or helium, caused a marked cytosolic acidification. Pharmacologic induction of intracellular acidification to similar levels reproduced the inhibitory effect. In vitro studies showed that CO2 insufflation lowered tissue pH and peritoneal macrophage LPS-stimulated TNF production. CONCLUSIONS The authors propose that cellular acidification induced by peritoneal CO2 insufflation contributes to blunting of the local inflammatory response during laparoscopic surgery.

Mechanisms of reprogrammed macrophage endotoxin signal transduction after lipopolysaccharide pretreatment

BACKGROUND Dysregulation of macrophage tumor necrosis factor (TNF) and interleukin-(IL-1) release results from repetitive lipopolysacharride (LPS) stimulation. In this study we investigated the mechanisms of LPS pretreatment (LPSp) signal transduction producing altered LPS-activated (LPSa) cytokine release. METHODS Murine macrophages were treated with medium alone, actinomycin D, cycloheximide, a protein kinase C inhibitor (H7), or the nitric oxide synthase inhibitor L-NMA. Macrophages were then pretreated with 100 ng/ml LPSp and cultured in medium alone, a nitric oxide donor (sodium nitroprusside), or a cyclic adenosine monophosphate donor (8 bromoadenosine) for 20 hours. Cultures were then washed, and fresh medium containing 1 microgram/ml LPSa was added. TNF and IL-1 release in 24-hour supernatant was measured by bioassays. RESULTS LPSp inhibited TNF and enhanced IL-1 release. The results with actinomycin D and cycloheximide suggested that LPSp effects did not require transcription, but IL-1 enhancement required protein synthesis. Addition of 8-bromo-cyclic adenosine monophosphate, H7, or nitroprusside prevented LPSp-induced augmentation of IL-1 but had no effect on inhibition of TNF release. Inhibition of LPSp-induced nitric oxide production with L-NMA had no effect on TNF or IL-1. CONCLUSIONS Complex, independent, but incompletely understood signal transduction pathways for LPSp-induced alterations in LPSa-stimulated macrophage TNF and IL-1 release were shown. Understanding altered signal transduction from prior LPS stimulation may suggest new therapies to control dysregulated macrophage cytokine release in sepsis.

Protein kinase C regulates macrophage tumor necrosis factor secretion: Direct protein kinase C activation restores tumor necrosis factor production in endotoxin tolerance

BACKGROUND Macrophages pretreated in vitro with endotoxin (LPSp) secrete less tumor necrosis factor (TNF) in response to a second LPS activating (LPSa) stimulus. Protein kinase C (PKC) is required for TNF secretion in a macrophage stimulated with LPSa. In these experiments we examined the role of PKC in TNF signal transduction in naive and tolerant macrophages. METHODS Murine macrophages were cultured +/- LPSp for 24 hours. Cultures were washed and treated for 1 hour with PKC inhibitors or phorbol myristate acetate (PMA), a direct PKC activator. Cells were then stimulated with a range of LPSa for 6 hours, and TNF was determined by bioassay. RESULTS LPSa-stimulated TNF secretion by nontolerant macrophages was inhibited by LPSp in the absence of PMA. PKC inhibitors decreased TNF by naive macrophages and exaggerated inhibition in tolerant cells. Depletion of PKC by 24 hours of PMA decreased TNF production by both naive and tolerant macrophages. PKC activation with PMA 1 hour before LPSa augmented TNF secretion in naive cells and reversed TNF inhibition of tolerant cells. CONCLUSIONS Direct PKC activation with PMA restored TNF secretion in LPS-tolerant macrophages. Endotoxin tolerance may alter the LPSa signal transduction pathway between the LPS receptor and PKC activation.

Inhibitory Kbα Control Of Nuclear Factor-kb Is Dysregulated In Endotoxin Tolerant Macrophages

The transcription factor nuclear factor (NF)-kappaB is thought to be required for endotoxin-stimulated tumor necrosis factor (TNF) and interleukin (IL)-1 gene transcription. Nuclear translocation of NF-kappaB is regulated by the cytoplasmic inhibitory factor IkappaBalpha. Low-dose lipopolysaccharide (LPS) pretreatment modulates cytokine release by altering subsequent LPS-activated signal transduction pathways. In this study, we examined the effect of LPS pretreatment exposure on IkappaBalpha and NF-kappaB following activation with LPS. Murine macrophages (Mphi were exposed to a range of LPS concentrations +/-24 h PreRx with 10 ng/mL LPS pretreatment. Cytoplasmic IkappaBalpha (Western immunoblot) and NF-kappaB (gel-shift assay) were assayed 30 min after LPS activation. Gene transcription for TNF was measured 6 h after LPS activation using RT-PCR. In the absence of LPS pretreatment, IkappaBalpha disappeared from the cytoplasm coincident with nuclear translocation of NF-kappaB. Tolerant Mphi had markedly enhanced levels of IkappaBalpha and normal to increased levels of NF-kappaB translocation with a different electrophoretic shift. LPS activation enhanced cytokine gene transcription in a dose-dependent manner, and this was unaltered by LPS pretreatment. Endotoxin-tolerant Mphi also had increased cytoplasmic levels of the p65 subunit of NF-kappaB. LPS tolerance is associated with increases of cytoplasmic IkappaBalpha p65, as well as enhanced NF-kappaB. We conclude that control of NF-kappaB translocation by IkappaBalpha is dysregulated in endotoxin-tolerant Mphi.

Defective lipopolysaccharide-dependent ERK 1/2 activation in endotoxin tolerant murine macrophages is reversed by direct protein kinase C stimulation.

Lipopolysaccharide (LPSp) pretreatment inhibits TNF secretion in endotoxin-tolerant macrophages via alterations in signal transduction pathways of LPS activation (LPSa). Protein kinase C inhibitors prevent TNF release in response to LPSa and direct protein kinase C activation with phorbol myristate acetate (PMA) restores TNF secretion after LPSp. In the current experiments the effect of protein kinase C modulation on LPSa-stimulated ERK 1/2 activation was investigated. Murine macrophage TNF production was determined after stimulation with 100 ng/mL of LPSa, +/- 24 h pretreatment with 10 ng/mL of LPSp. Direct protein kinase C activators (PMA or indolactam) or inhibitors (H7 or bisindolylmaleimide) were added 1 h before LPSa. Diphosphorylated ERK 1/2 was assayed after LPSa stimulation by Western blot. LPS tolerance after LPSp was characterized by inhibition of LPSa-stimulated TNF and accompanied by impaired ERK 1/2 activation by LPSa. Protein kinase C activation with PMA or indolactam restored ERK 1/2 activation and TNF secretion. Inhibition of protein kinase C with H7 or bisindolylmaleimide prevented TNF secretion and ERK 1/2 activation by LPSa. These findings suggest that both ERK 1/2 and protein kinase C are required for TNF production in nontolerant macrophages and that LPS tolerance may be associated with an inability to phosphorylate ERK 1/2.

Lipopolysaccharide pretreatment produces macrophage endotoxin tolerance via a serum-independent pathway

BACKGROUND Lipopolysaccharide activation (LPSa) of macrophages is thought to occur via a CD14-dependent mechanism with a requirement for the serum factor, lipopolysaccharide binding protein. LPS-stimulated, CD14-dependent signal transduction is associated with phosphorylation of mitogen-activated protein kinase (MAPK), nuclear factor-kappaB (NF-kappaB) translocation, and secretion of tumor necrosis factor (TNF) and interleukin-1 (IL-1). Macrophage endotoxin tolerance after low-dose LPS pretreatment (LPSp) is characterized by inhibition of LPSa-stimulated TNF and augmentation of IL-1 secretion. We sought to determine the role of CD14-dependent pathways in the induction of endotoxin tolerance by comparing the effects of LPSp in the presence or absence of serum. METHODS Murine peritoneal macrophages were exposed to a range of LPSp concentrations in the presence or absence of serum. MAPK activation and NF-kappaB were assayed 30 minutes after LPSp stimulation. TNF production and IL-1 were measured 6 hours after stimulation with 100 ng/mL LPSa, with or without 24-hour 10 ng/mL LPSp. RESULTS In the presence of serum, 100 ng/mL LPSp activated MAPK and NF-kappaB, whereas no activation of MAPK or NF-kappaB was seen at this LPSp concentration in the absence of serum. The absence of serum during 10 ng/mL LPSp did not prevent LPSp-mediated inhibition of TNF secretion, and it significantly augmented IL-1 secretion after stimulation with 100 ng/mL LPSa in the presence of serum. CONCLUSION Induction of the alterations in subsequent LPSa-stimulated cytokine secretion characteristic of endotoxin tolerance by LPSp occurs via a serum-independent signal transduction pathway.

Acetazolamide treatment prevents in vitro endotoxin-stimulated tumor necrosis factor release in mouse macrophages.

We previously showed that incubation in carbon dioxide (CO2), but not air or helium (He), markedly decreased macrophage intracellular pH (pH1) and resulted in reversible inhibition of lipopolysaccharide- (LPS) stimulated tumor necrosis factor (TNF) and interleukin-1 release. We sought to determine whether carbonic anhydrase inhibition with acetazolamide would prevent CO2-mediated inhibition of LPS-stimulated TNF release. Murine peritoneal macrophages were treated with acetazolamide for 1 h under control atmosphere (95% air/5% CO2) and then switched to incubator modules containing: 1) 80% CO2/20% O2, 2) 80% He/20% O2, or 3) 100% air. Before transfer to experimental atmospheric conditions the macrophages were stimulated with 0 or 1 μg/mL of LPS (Escherichia coli 0111B4). Supernatant TNF was measured 4 h later by bioassay. In parallel experiments LPS-stimulated cytokine mRNA was estimated using reverse transcriptase polymerase chain reaction (RT-PCR) 2 h after LPSstimulated. Viability was determined using dye uptake. Incubation in CO2 or helium had no effect on TNF production in the absence of LPS. In the absence of acetazolamide CO2 produced marked inhibition of LPS-stimulated TNF release, but this was not blocked by the presence of acetazolamide. This CO2-mediated inhibition of TNF was associated with normal levels of TNF mRNA. In acetazolamide-treated macrophages, LPS resulted in a dose-dependent inhibition of TNF release, but not TNF mRNA induction by LPS. Factors that alter intracellular pH regulation may modulate LPS-stimulated cytokine production.