LPS-tolerant Macrophages Research Articles

Less Severe Lipopolysaccharide-Induced Inflammation in Conditional mgmt-Deleted Mice with LysM-Cre System: The Loss of DNA Repair in Macrophages.

Despite the known influence of DNA methylation from lipopolysaccharide (LPS) activation, data on the O6-methylguanine-DNA methyltransferase (MGMT, a DNA suicide repair enzyme) in macrophages is still lacking. The transcriptomic profiling of epigenetic enzymes from wild-type macrophages after single and double LPS stimulation, representing acute inflammation and LPS tolerance, respectively, was performed. Small interfering RNA (siRNA) silencing of mgmt in the macrophage cell line (RAW264.7) and mgmt null (mgmtflox/flox; LysM-Crecre/-) macrophages demonstrated lower secretion of TNF-α and IL-6 and lower expression of pro-inflammatory genes (iNOS and IL-1β) compared with the control. Macrophage injury after a single LPS dose and LPS tolerance was demonstrated by reduced cell viability and increased oxidative stress (dihydroethidium) compared with the activated macrophages from littermate control mice (mgmtflox/flox; LysM-Cre-/-). Additionally, a single LPS dose and LPS tolerance also caused mitochondrial toxicity, as indicated by reduced maximal respiratory capacity (extracellular flux analysis) in the macrophages of both mgmt null and control mice. However, LPS upregulated mgmt only in LPS-tolerant macrophages but not after the single LPS stimulation. In mice, the mgmt null group demonstrated lower serum TNF-α, IL-6, and IL-10 than control mice after either single or double LPS stimulation. Suppressed cytokine production resulting from an absence of mgmt in macrophages caused less severe LPS-induced inflammation but might worsen LPS tolerance.

Endotoxin tolerance induced by Porphyromonas gingivalis lipopolysaccharide alters macrophage polarization

Endotoxin tolerance refers to a state refractory to subsequent lipopolysaccharide (LPS) stimulations following a primary LPS exposure. To study the relationship between endotoxin tolerance and macrophage polarization, endotoxin tolerance was induced by 1 μg/mL LPS from the periodontal pathogen, Porphyromonas gingivalis (P. gingivalis), in peritoneal macrophages (PMs) and bone marrow-derived macrophages (BMDMs). Repeated P. gingivalis LPS challenges increased the quantities of CD206+ PMs, while the number of CD86+CD206+ PMs was reduced compared with the non-tolerant group (p < 0.05). However, there were no changes in BMDMs (p > 0.05). Down regulations of TNF-α, IL-12, nitric oxide and MMP-2 production, and upregulated IL-10, MMP-9 levels and arginase-1 activities occurred in tolerant PMs and BMDMs (p < 0.05). P. gingivalis LPS-tolerant PMs and BMDMs also enhanced scrape-wound healing abilities of 15p-1 cells (p < 0.05). Expressions of phospho-signal transducer and activator of transcription 6 (p-STAT6) and protein tyrosine phosphatase 1B (PTP1B) were increased, while p-MEK1/2 levels were downregulated in tolerant PMs and BMDMs (p < 0.05). IL-10 production in tolerant Stat6 knockdown RAW264.7 cells was lower than tolerant control cells (p < 0.05). P. gingivalis LPS-tolerant macrophages represented an intermediate state between M1/M2 polarization, which functioned as M2-like cells, and led to limited inflammatory responses and enhanced wound healing activities. The PTP1B-MEK1/2-STAT6 signaling pathway might be involved in the polarization of tolerant macrophages.

The chemotherapeutic drug carboplatin affects macrophage responses to LPS and LPS tolerance via epigenetic modifications

Following re-exposure to lipopolysaccharide (LPS), macrophages exhibit an immunosuppressive state known as LPS tolerance, which is characterized by repressed proinflammatory cytokine production. LPS-induced tolerance in macrophages is mediated in part by epigenetic changes. Carboplatin, an anticancer chemotherapeutic drug, exerts its effect by inhibiting DNA replication and transcription, as well as through epigenetic modifications. Through an unbiased screen, we found that carboplatin rescued TNF-α and IL-6 production in LPS-tolerant macrophages. Transcriptomic analysis and gene set enrichment analyses revealed that p53 was one of the most significantly upregulated hallmarks in both LPS-primed and LPS-tolerant macrophages in the presence of carboplatin, while E2F and G2/M were the most negatively regulated hallmarks. Heterochromatin protein 1 (HP1-α), which is associated with gene silencing, was significantly reduced in carboplatin-treated LPS-tolerant macrophages at the mRNA and protein levels. Dynamic changes in the mRNA level of genes encoding H3K9me3 methyltransferases, setdb2, kdm4d, and suv39h1 were induced in the presence of carboplatin in LPS-tolerant macrophages. Taken together, we provide evidence that carboplatin treatment interferes with proinflammatory cytokine production during the acute LPS response and LPS tolerance in macrophages, possibly via H3K9me3 modification.

LPS Tolerance Inhibits Cellular Respiration and Induces Global Changes in the Macrophage Secretome

Inflammatory response plays an essential role in the resolution of infections. However, inflammation can be detrimental to an organism and cause irreparable damage. For example, during sepsis, a cytokine storm can lead to multiple organ failures and often results in death. One of the strongest triggers of the inflammatory response is bacterial lipopolysaccharides (LPS), acting mostly through Toll-like receptor 4 (TLR4). Paradoxically, while exposure to LPS triggers a robust inflammatory response, repeated or prolonged exposure to LPS can induce a state of endotoxin tolerance, a phenomenon where macrophages and monocytes do not respond to new endotoxin challenges, and it is often associated with secondary infections and negative outcomes. The cellular mechanisms regulating this phenomenon remain elusive. We used metabolic measurements to confirm differences in the cellular metabolism of naïve macrophages and that of macrophages responding to LPS stimulation or those in the LPS-tolerant state. In parallel, we performed an unbiased secretome survey using quantitative mass spectrometry during the induction of LPS tolerance, creating the first comprehensive secretome profile of endotoxin-tolerant cells. The secretome changes confirmed that LPS-tolerant macrophages have significantly decreased cellular metabolism and that the proteins secreted by LPS-tolerant macrophages have a strong association with cell survival, protein metabolism, and the metabolism of reactive oxygen species.

Artesunate interacts with the vitamin D receptor to reverse sepsis-induced immunosuppression in a mouse model via enhancing autophagy.

Immunosuppression is the predominant cause of mortality for sepsis because of failure to eradicate pathogens. No effective and specific drugs capable of reversing immunosuppression are clinically available. Evidences implicate the involvement of the vitamin D receptor (NR1I1) in sepsis-induced immunosuppression. The anti-malarial artesunate was investigated to determine action on sepsis-induced immunosuppression. The effect of artesunate on sepsis-induced immunosuppression was investigated in mice and human and mice cell lines. Bioinformatics predicted vitamin D receptor as a candidate target for artesunate, which was then identified using PCR and immunoblotting. Vdr, Atg16l1 and NF-κB p65 were modified to investigate artesunate 's effect on pro-inflammatory cytokines release, bacterial clearance and autophagy activities in sepsis-induced immunosuppression. Artesunate significantly reduced the mortality of caecal ligation and puncture (CLP)-induced sepsis immunosuppression mice challenged with Pseudomonas aeruginosa and enhanced pro-inflammatory cytokine release and bacterial clearance to reverse sepsis-induced immunosuppression in vivo and in vitro. Mechanistically, artesunate interacted with vitamin D receptor, inhibiting its nuclear translocation, which influenced ATG16L1 transcription and subsequent autophagy activity. Artesunate inhibited the physical interaction between vitamin D receptor and NF-κB p65 in LPS-tolerant macrophages and then promoted the nuclear translocation of NF-κB p65, which activated the transcription of NF-κB p65 target genes such as pro-inflammatory cytokines. Our findings provide evidence that artesunate interacted with vitamin D receptor to reverse sepsis-induced immunosuppression in an autophagy and NF-κB-dependent manner, highlighting a novel approach for sepsis treatment and drug repurposing of artesunate has a bidirectional immunomodulator.

Dysregulation of Lipid Metabolism in Macrophages Is Responsible for Severe Endotoxin Tolerance in FcgRIIB-Deficient Lupus Mice.

FcgRIIB dysfunction is commonly found in patients with lupus, especially in Asia. LPS-tolerance is prominent in FcgRIIB–/– lupus mice. LPS-tolerant macrophages demonstrate cell energy depletion, which might affect lipid metabolism. Therefore, to explore lipid metabolism, LPS-tolerance was induced twice by LPS administration in macrophages and in mice. LPS-tolerant FcgRIIB–/– macrophages demonstrated lesser mitochondrial DNA (mtDNA), more severe ATP depletion, lower cytokine production, and higher lipid accumulation (oil red O staining) compared to LPS-tolerant WT cells. Mass-spectrometry-based lipidomic analysis demonstrated a higher abundance of phosphatidylethanolamine (PE) phospholipid in LPS-tolerant FcgRIIB–/– macrophages than WT cells. This was at least in part due to the lower expression of phosphatidylethanolamine N-methyltransferase (pemt), an enzyme that converts PE to phosphatidylcholine (PC). Aminoimidazole-4-carboxamide ribonucleotide (AICAR), a pemt inhibitor, worsens LPS-tolerance in WT macrophages and supports the impact of pemt upon LPS-tolerant FcgRIIB–/– macrophages. Additionally, phosphorylated AMP-activated protein kinase (AMPK-p), a molecule for ATP-restoration associated with pemt, and phosphorylated acetyl CoA carboxylase, a downstream signaling of AMPK-p, were higher in LPS-tolerant FcgRIIB–/– macrophages than WT. Furthermore, Compound C, an AMPK inhibitor, attenuated LPS-tolerance in both FcgRIIB–/– macrophages and mice. Taken together, the intense decrease in cytokine production after the second LPS stimulation (LPS-tolerance) in FcgRIIB–/– macrophages was possibly due to the impact of an immense cytokine synthesis after the first dose of LPS. This includes using up PEMT, an enzyme of phospholipid synthesis during cytokine production, and AMPK-p induction in response to profound ATP-depletion. Therefore, the manipulation of the AMPK/PEMT axis provides a novel therapeutic candidate for the treatment of severe LPS-tolerance in lupus.

Glucocorticoid-induced leucine zipper: a critical factor in macrophage endotoxin tolerance.

Induction of glucocorticoid-induced leucine zipper (GILZ) by glucocorticoids plays a key role in their anti-inflammatory action. In activated macrophages, GILZ levels are downregulated via tristetraprolin-mediated GILZ mRNA destabilization. To assess the functional significance of GILZ downregulation, we generated myeloid-specific GILZ knockout (KO) mice. GILZ-deficient macrophages displayed a higher responsiveness toward LPS, as indicated by increased TNF-α and IL-1β expression. This effect was due to an activation of ERK, which was significantly amplified in GILZ KO cells. The LPS-induced activation of macrophages is attenuated upon pretreatment of macrophages with low-dose LPS, an effect termed endotoxin tolerance. In LPS-tolerant macrophages, GILZ mRNA was stabilized, whereas ERK activation was strongly decreased. In contrast, GILZ KO macrophages exhibited a strongly reduced desensitization. To explore the contribution of GILZ expression in macrophages to endotoxin tolerance in vivo, we treated GILZ KO mice with repeated i.p. injections of low-dose LPS followed by treatment with high-dose LPS. LPS pretreatment resulted in reduced proinflammatory mediator expression upon high-dose LPS treatment in serum and tissues. In contrast, cytokine induction was preserved in tolerized GILZ KO animals. In summary, our data suggest that GILZ is a key regulator of macrophage functions.

Gαi1 and Gαi3 regulate macrophage polarization by forming a complex containing CD14 and Gab1

Heterotrimeric G proteins have been implicated in Toll-like receptor 4 (TLR4) signaling in macrophages and endothelial cells. However, whether guanine nucleotide-binding protein G(i) subunit alpha-1 and alpha-3 (Gαi1/3) are required for LPS responses remains unclear, and if so, the underlying mechanisms need to be studied. In this study, we demonstrated that, in response to LPS, Gαi1/3 form complexes containing the pattern recognition receptor (PRR) CD14 and growth factor receptor binding 2 (Grb2)-associated binding protein (Gab1), which are required for activation of PI3K-Akt signaling. Gαi1/3 deficiency decreased LPS-induced TLR4 endocytosis, which was associated with decreased phosphorylation of IFN regulatory factor 3 (IRF3). Gαi1/3 knockdown in bone marrow-derived macrophage cells (Gαi1/3 KD BMDMs) exhibited an M2-like phenotype with significantly suppressed production of TNF-α, IL-6, IL-12, and NO in response to LPS. The altered polarization coincided with decreased Akt activation. Further, Gαi1/3 deficiency caused LPS tolerance in mice. In vitro studies revealed that, in LPS-tolerant macrophages, Gαi1/3 were down-regulated partially by the proteasome pathway. Collectively, the present findings demonstrated that Gαi1/3 can interact with CD14/Gab1, which modulates macrophage polarization in vitro and in vivo.

Identification of a Unique Hybrid Macrophage-Polarization State following Recovery from Lipopolysaccharide Tolerance

LPS tolerance is an essential immune-homeostatic response to repeated exposure to LPS that prevents excessive inflammatory responses. LPS tolerance induces a state of altered responsiveness in macrophages, resulting in repression of proinflammatory gene expression and increased expression of factors that mediate the resolution of inflammation. In this study, we analyzed the transcriptional plasticity of macrophages following LPS tolerance using genome-wide transcriptional profiling. We demonstrate that LPS tolerance is a transient state and that the expression of proinflammatory genes is restored to levels comparable to the acute response to LPS. However, following recovery from LPS tolerance a number of genes remained locked in a tolerizable state, including IL-33, CD86, IL-10, and NFIL3. Furthermore, we identified of a number of genes uniquely induced following recovery from LPS tolerance. Thus, macrophages adopt a unique transcriptional profile following recovery from LPS tolerance and have a distinct expression pattern of regulators of Ag presentation, antiviral responses, and transcription factors. Our data suggest that recovery from LPS tolerance leads to a hybrid macrophage activation state that is proinflammatory and microbicidal in nature but that possesses a regulatory anti-inflammatory profile distinct from that of LPS-tolerant and LPS-activated macrophages.

RIP140, a Janus metabolic switch involved in defense functions

RIP140, a Janus metabolic switch involved in defense functions

Regulation of LPS Tolerance in Macrophages (48.2)

Abstract Lipopolysaccharide (LPS) tolerance is an essential immune-homeostatic response which prevents unnecessary over-activation of the inflammatory response. LPS tolerance induces a state of reduced responsiveness to repeated challenges with LPS, resulting in decreased expression of pro-inflammatory modulators, up-regulation of antimicrobials and other mediators promoting the resolution of inflammation. LPS tolerance is a transient state and LPS tolerant macrophages (MΦT) return to an LPS responsive state within days of initial stimulation. Macrophages which have recovered from a tolerant state (MΦR) remain largely uncharacterised. Gene expression profiling reveals that LPS tolerance recovery triggers an extensive modulation of the gene expression profile of macrophages distinct from that of (MΦT) and LPS activated macrophages (MΦA). Recovery from LPS tolerance at 88 hours post treatment leads to expression of key pro-inflammatory genes at levels comparable to MΦA. MΦR show increased expression of key modulators of the immune response not previously observed during classical activation. We propose that MΦR represent a novel macrophage phenotype. Furthermore we propose that the recovery from LPS tolerance promotes a global modulation of gene expression with distinct expression profiles between genes promoting inflammation and genes key to the resolution of inflammation. In addition we present an expression analysis of NF-κB and its regulators in naïve, MΦT and MΦR cells.

내독소내성 마우스에서 Nitric Oxide 생성에 미치는 림프구 부전

본 연구에서는 내독소내성 상태에 있는 마우스 세포의 싸이토카인 생성능을 측정하기 위하여 대식세포-림프구 공동배양계를 이용한 nitric oxide (NO) 생성을 조사하였다. 마우스 복강 대식세포에 lipopolysaccharide (LPS)와 interferon-g (IFN-g)를 처리시 NO 생성이 증가되었으며, tumor necrosis factor-a (TNF-a) 또한 LPS처럼 NO 합성을 자극하는 것을 관찰할 수 있었다. 한편, 대식세포를 비장세포와 공동배양시, LPS 단독처리만으로도 NO 합성이 증가되었다. 반면, 2.5 mg/kg LPS로 전처리하고 치사량의 LPS를 2차 투여한 마우스의 경우, 마우스의 치사 및 혈중 TNF-a와 IFN-g가 증가되지 않았다. 또한 LPS-내성 마우스로부터 분리한 대식세포를 정상 비장세포와 공동배양시 LPS에 의한 NO생성이 일어나지 않았으며, 외래 TNF-a에 의한 NO 생성도 일어나지 않았다. 이와 아울러 정상 대식세포와 LPS 내성 마우스로부터 분리한 비장세포를 공동배양하였을 때, LPS 자극으로 인한 NO 생성이 일어나지 않았으며, 이러한 억제현상은 외래 IFN-g 또는 IFN-g 생성을 촉진시키는 concanavalin A (ConA)에 의해서 다시 역전되었다. 이러한 결과는 대식세포 뿐만 아니라 림프구도 LPS 내성에 관여하는 것을 보여준다고 사료된다. INF-g는 TNF-a 발현을 증가시키기 때문에, 림프구의 INF-g 합성 감소는 LPS에 내성을 보이는 대식세포의 TNF-a 합성 저하와 상호작용으로 내독소내성 상태를 유도하며 과도한 염증반응을 억제하는 것으로 사료된다. 따라서 LPS 내독소내성은 중환자의 심각한 패혈증에 대한 예방법으로 활용될 수 있을 것으로 기대된다. In this study, nitric oxide (NO) production in a macrophage-lymphocyte co-culture system was used to assess the cytokine producing capability of cells during endotoxin tolerance in mice. Incubation of peritoneal macrophages with interferon-<TEX>$\tau$</TEX> (IFN-<TEX>$\tau$</TEX>) in the presence of lipopolysaccharide (LPS) augmented NO synthesis. Exogenous tumor necrosis factor-<TEX>$\alpha$</TEX>(TNF-<TEX>$\alpha$</TEX>) could also replace LPS for the stimulation of NO production. Macrophages co-cultured with splenic lymphocytes showed augmented NO synthesis by LPS alone. However, pretreatment of mice with 2.5 mg/kg LPS completely prevented the lethality and the increase of blood TNF-<TEX>$\alpha$</TEX> and IFN-<TEX>$\tau$</TEX> after the second challenge with a lethal dose of LPS. In addition, when macrophages prepared from LPS-tolerant mice were co-cultured with normal splenocytes, LPS also could not induce the production of NO, even in the presence of exogenous TNF-<TEX>$\alpha$</TEX>. Moreover, when normal macrophages were co-cultured with splenocytes obtained from LPS-tolerant mice, stimulation with LPS could not evoke the NO production enhancement. However, this down-regulation was able to reverse by exogenous IFN-<TEX>$\tau$</TEX> or concanavalin A (ConA), a stimulator of IFN-<TEX>$\tau$</TEX> production. Our results indicate that not only macrophages but also lymphocytes contribute to LPS tolerance. As INF-<TEX>$\tau$</TEX> can enhance the expression of TNF-<TEX>$\alpha$</TEX>, the decrease of INF-<TEX>$\tau$</TEX>synthesis from lymphocytes may orchestrate with the decrease of TNF-<TEX>$\alpha$</TEX> synthesis from LPS-tolerant macrophages for the production of tolerant state and the prevention of excessive inflammation. Therefore, LPS tolerance may be exploited for prophylaxis of severe sepsis in patients at risk.

A genome-wide analysis of LPS tolerance in macrophages

Among the multiple mechanisms that control the intensity and duration of macrophage activation, the development of a state of refractoriness to a second stimulation in cells treated with lipopolysaccharide (LPS) has long been recognized. Release of inhibitory cytokines and alterations in intracellular signaling pathways may be involved in the development of LPS tolerance. Although a number of molecules have been implicated, a detailed picture of the molecular changes in LPS tolerance is still missing. We have used a genome-wide gene expression analysis approach to (i) define which fraction of LPS target genes are subject to tolerance induction and (ii) identify genes that are expressed at high levels in tolerant macrophages. Our data show that in LPS-tolerant macrophages the vast majority of LPS-induced gene expression is abrogated. The extent of tolerance induction varies for individual genes, and a small subset of genes appears to be exempted. Compared to other negative control mechanisms of macrophages, e.g., IL-10-induced deactivation, LPS tolerance inhibits a much wider range of transcriptional targets. Some previously described negative regulators of TLR signaling (e.g. IRAK-M) were confirmed as expressed at higher levels in LPS-tolerant macrophages. In addition, we discuss other potential players in LPS tolerance identified in this group of genes.

Cellular mechanisms that cause suppressed gamma interferon secretion in endotoxin-tolerant mice.

Endotoxin (lipopolysaccharide [LPS]) tolerance is a state of altered immunity characterized, in part, by suppression of LPS-induced gamma interferon (IFN-gamma) expression. However, the cellular mediators regulating LPS-induced production of IFN-gamma in normal mice and the effect of LPS tolerance on these mediators has not been well characterized. Our studies show that macrophage dysfunction is the primary factor causing suppressed IFN-gamma expression in LPS-tolerant mice. Specifically, LPS-tolerant macrophages have a markedly impaired ability to induce IFN-gamma secretion by T cells and NK cells obtained from either control or LPS-tolerant mice. However, T cells and NK cells isolated from LPS-tolerant mice produce normal levels of IFN-gamma when cocultured with control macrophages or exogenous IFN-gamma-inducing factors. Assessment of important IFN-gamma-regulating factors showed that interleukin-12 (IL-12) and costimulatory signals provided by IL-15, IL-18, and CD86 are largely responsible for LPS-induced IFN-gamma expression in control mice. IL-10 is an inhibitor of IFN-gamma production in both the control and LPS-tolerant groups. Expression of IL-12 and the IL-12 receptor beta1 (IL-12Rbeta1) and IL-12Rbeta2 subunits are suppressed in the spleens of LPS-tolerant mice. LPS-tolerant splenocytes also exhibit decreased production of IL-15 and IL-15Ralpha. However, expression of IL-18 and the B7 proteins CD80 and CD86 are unchanged or increased compared to controls after induction of LPS tolerance. CD28, a major receptor for B7 proteins, is also increased in the spleens of LPS-tolerant mice. Expression of the inhibitory cytokine IL-10 and the IL-10R are sustained after induction of LPS tolerance. These data show that suppression of IFN-gamma production in LPS-tolerant mice is largely due to macrophage dysfunction and provide insight into the cellular alterations that occur in LPS tolerance. This study also better defines the factors that mediate LPS-induced IFN-gamma production in normal mice.

Lipopolysaccharide tolerance in murine peritoneal macrophages induces downregulation of the lipopolysaccharide signal transduction pathway through mitogen-activated protein kinase and nuclear factor-κB cascades, but not lipopolysaccharide-incorporation steps

Endotoxin/lipopolysaccharide (LPS) tolerance, a hyporesponsive state to endotoxin or LPS stimulation, was induced in murine peritoneal macrophages by previous exposure of macrophages to LPS. Expression of tumor necrosis factor (TNF)-α and interleukin (IL)-6 mRNA in response to LPS stimulation was suppressed in LPS-tolerant macrophages. Tyrosine phosphorylations in response to LPS of 40–45-kDa proteins in non-tolerant macrophages were also suppressed in LPS-tolerant macrophages. These proteins corresponded to two members of the mitogen-activated protein kinase (MAPK) family, ERK and p38. In addition to these proteins, another MAPK family protein, JNK, was also suppressed in LPS-tolerant macrophages. Activation of Raf-1, located in the upstream portion of ERK cascades, was also suppressed by LPS-tolerance induction. These suppressions in LPS-tolerant macrophages were exhibited against stimulation by an LPS agonist like taxol, but not towards stimulation by an unrelated activator like phorbol ester (PMA). Activation of the transcription factor NF-κB, which is supposed to be one of the components of another important pathway for transduction of LPS-stimulated cytokine producing signals, was strongly suppressed and degradation of IκB, an inhibitor of NF-κB, was also severely diminished in LPS-tolerant macrophages. Although a monosaccharide lipid A analog, GLA-58, was able to stimulate macrophages to activate ERK proteins without cytokine production, pretreatment of macrophages with this compound suppressed both LPS-stimulated activation of ERK and cytokine production. Furthermore, downregulation of LPS-uptake in LPS-tolerant macrophages was not observed. Based on all these findings, LPS tolerance might be caused by the previous activation of some components on LPS-signaling pathways. This may then induce a refractory state in key LPS-signal transducer molecules located downstream of the cell membrane LPS receptor and upstream of the branching point in intracellular cascades for activation of MAPK and NF-κB, probably in some initial steps of intracellular signaling.

Lipopolysaccharide- and paclitaxel (Taxol)-induced tolerance in murine peritoneal macrophages

LPS-tolerance, a state of refractoriness to LPS-stimulation, is induced in murine peritoneal macrophages by prior exposure to LPS. LPS-induced expression of TNF and IL-6 mRNA as well as activation of various intracellular kinases and factors, including ERK, p38, JNK, Raf-1 and NF-κB were all suppressed in LPS-tolerant macrophages; responses to stimulation by paclitaxel (Taxol™), an LPS agonist, were similarly suppressed, but responses to phorbol esters (PMA) were unaffected. Binding and uptake of [125I]-labeled LPS to tolerant macrophages was somewhat greater in tolerant than in non-tolerant macrophages. Thus, the refractory state appears to involve inhibition or blockade of LPS-signaling molecules located downstream of the cell membrane LPS receptor and upstream of the branch point in the intracellular cascades leading to activation of MAPK and NFκB. LPS conditioning also suppressed LPS- and Taxol-induced TNF production, but augmented nitric oxide (NO) production. In contrast, Taxol conditioning failed to suppress LPS-induced TNF production. Conditioning with the synthetic taxoid analog, nor-seco-taxoid, which does not induce macrophage activation, enhanced LPS- and Taxol-induced NO production. These findings provide us with new information about the relationship between the LPS and Taxol receptors as well as about the signaling pathways leading to TNF and NO production.

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 (LPS p) secrete less tumor necrosis factor (TNF) in response to a second LPS activating (LPS a) stimulus. Protein kinase C (PKC) is required for TNF secretion in a macrophage stimulated with LPS a. In these experiments we examined the role of PKC in TNF signal transduction in naive and tolerant macrophages. Methods. Murine macrophages were cultured ± LPS p 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 LPS a for 6 hours, and TNF was determined by bioassay. Results. LPS a-stimulated TNF secretion by nontolerant macrophages was inhibited by LPS p 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 LPS a 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 LPS a signal transduction pathway between the LPS receptor and PKC activation.

Independent Signal Transduction Pathways for IL-1 and TNF in LPS-Tolerant Macrophages

Macrophages rendered "tolerant" by pretreatment with low-dose endotoxin (LPS p) release less TNF and more IL-1 in response to a second activating endotoxin exposure (LPS a). We hypothesized that LPS p pretreatment alters signal transduction pathways for TNF and IL-1 independently. The effect of pretreatment with LPS p alone was compared to pretreatment with LPS p plus defined second-messenger pathway agonists or antagonists. Murine peritoneal macrophages were pretreated in vitro for 4 hr with LPS p or PMA or LPS p plus protein kinase C inhibitor (PKCi) or 8-bromo-cAMP. Cells were then washed and cultured with medium alone for 20 hr. Macrophages were also pretreated with LPS p plus indomethacin for the total 24-hr pretreatment interval. Cells were then stimulated for 24 hr with LPS a, after which supernatant TNF and IL-1 were measured by bioassay. In the absence of LPS p, mediators were increased by LPS a in a dose-dependent manner. LPS p pretreatment inhibited TNF and augmented IL-1 in response to LPS a. Pretreatment with PMA partially reproduced LPS p pretreatment. Pretreatment with PKCi alone increased both TNF and IL-1 release by LPS a. The combination of LPS p plus PKCi pretreatment further enhanced IL-1 release without affecting TNF inhibition. The addition of indomethacin had a similar effect. The combination of LPS p plus 8-bromo-cAMP blocked the augmentation of IL-1 without changing TNF inhibition. Macrophage endotoxin tolerance following LPS p pretreatment alters LPS a-triggered TNF and IL-1 release by independent signal transduction pathways.