Short-term treatment of RAW264.7 macrophages with adiponectin increases tumor necrosis factor-alpha (TNF-alpha) expression via ERK1/2 activation and Egr-1 expression: role of TNF-alpha in adiponectin-stimulated interleukin-10 production

Adiponectin is an adipokine with potent anti-inflammatory properties. However, the mechanisms by which adiponectin suppresses macrophage function are not well understood. Treatment of RAW264.7 macrophages with adiponectin for 18 h decreased lipopolysaccharide (LPS)-stimulated tumor necrosis factor-alpha (TNF-alpha) production. Here we demonstrate that globular adiponectin (gAcrp) initially increased TNF-alpha expression in RAW264.7 macrophages; this TNF-alpha then contributed to increased expression of interleukin-10, which in turn was required for the development of tolerance to subsequent LPS exposure. gAcrp-mediated increases in TNF-alpha mRNA accumulation were associated with increased TNF-alpha promoter activity. gAcrp increased the DNA binding activity of both Egr-1 and NFkappaB; mutation of either the Egr-1 or NFkappaB binding sites in the TNF-alpha promoter decreased gAcrp-stimulated promoter activity. Further, co-transfection with either dominant negative Egr-1 or the IkappaB super-repressor prevented gAcrp-stimulated TNF-alpha promoter activity. gAcrp also increased Egr-1 promoter activity, mRNA accumulation, and DNA binding activity. Inhibition of ERK1/2 with U0126 potently suppressed gAcrp-stimulated Egr-1 promoter activity, as well as TNF-alpha promoter activity. In summary, these data demonstrate that adiponectin initially increases TNF-alpha production by macrophages via ERK1/2-->Egr-1 and NFkappaB-dependent mechanisms; these increases in TNF-alpha in turn lead to increased expression of interleukin-10 and an eventual dampening of LPS-mediated cytokine production in macrophages.     

Effects of adiponectin in TNF-α, IL-6, and IL-10 cytokine production from coronary artery disease macrophages

Adiponectin, an adipose tissue secreted protein, exhibits anti-inflammatory and antiatherogenic properties. We examined the effects of the globular and full-length adiponectin on cytokine production in macrophages derived from Coronary Artery Disease (CAD) patients and control individuals. Adiponectin's effects in human macrophages upon lipopolysaccharide (LPS) treatment were also examined. Full length adiponectin acted differently on TNF-α and IL-6 production by upregulating TNF-α and IL-6 protein production, but not their mRNA expression. Additionally, full length adiponectin was unable to abrogate LPS proinflammatory effect in TNF-α and IL-6 mRNA expression in CAD and NON-CAD macrophages. In contrast, globular adiponectin appeared to have proinflammatory properties by potently upregulating TNF-α and IL-6 mRNA and protein secretion in human macrophages while subsequently rendered cells resistant to further proinflammatory stimuli. Moreover, both forms of adiponectin powerfully suppressed scavenger MSR-AI mRNA expression and augmented IL-10 protein release, both occurring independently of the presence of LPS or CAD. These data indicate that adiponectin could potentially protect human macrophages via the elevated IL-10 secretion and the suppression of MSR-AI expression. It can also be protective in CAD patients since the reduced adiponectin-induced IL-6 release in CAD macrophages compared to controls, could be beneficial in the development of inflammation related atherosclerosis.     

Adiponectin induces TNF-alpha and IL-6 in macrophages and promotes tolerance to itself and other pro-inflammatory stimuli

Adiponectin exerts anti-inflammatory effects via macrophages, suppressing the production of pro-inflammatory cytokines in response to bacterial lipopolysaccharide (LPS). Here, we provide experimental evidence that the "anti-inflammatory" effect of adiponectin may be due to an induction of macrophage tolerance: globular adiponectin (gAd) is a powerful inducer of TNF-alpha and IL-6 secretion in primary human peripheral macrophages, in the THP-1 human macrophage cell line, and in primary mouse peritoneal macrophages. Pre-exposure of macrophages to 10 microg/ml gAd rendered them tolerant to further gAd exposure or to other pro-inflammatory stimuli such as TLR3 ligand polyI:C and TLR4 ligand LPS, while pre-exposure to 1 microg/ml of and re-exposure to 10 microg/ml gAd unmasked its pro-inflammatory properties. GAd induced NF-kappaB activation and tolerance to further gAd or LPS exposure. Our data suggest that adiponectin constant presence in the circulation in high levels (in lean subjects) renders macrophages resistant to pro-inflammatory stimuli, including its own.     

Adiponectin normalizes LPS-stimulated TNF-alpha production by rat Kupffer cells after chronic ethanol feeding

Chronic ethanol feeding sensitizes Kupffer cells to activation by lipopolysaccharide (LPS), leading to increased production of tumor necrosis factor-alpha (TNF-alpha). Adiponectin treatment protects mice from ethanol-induced liver injury. Because adiponectin has anti-inflammatory effects on macrophages, we hypothesized that adiponectin would normalize chronic ethanol-induced sensitization of Kupffer cells to LPS-mediated signals. Serum adiponectin concentrations were decreased by 45% in rats fed an ethanol-containing diet for 4 wk compared with pair-fed rats. Adiponectin dose dependently inhibited LPS-stimulated accumulation of TNF-alpha mRNA and peptide in Kupffer cells from both pair- and ethanol-fed rats. Kupffer cells from ethanol-fed rats were more sensitive to both globular (gAcrp) and full-length adiponectin (flAcrp) than Kupffer cells from pair-fed controls with suppression at 10 ng/ml adiponectin after chronic ethanol feeding. Kupffer cells expressed both adiponectin receptors 1 and 2; chronic ethanol feeding did not change the expression of adiponectin receptor mRNA or protein. gAcrp suppressed LPS-stimulated ERK1/2 and p38 phosphorylation as well as IkappaB degradation at 100-1,000 ng/ml in Kupffer cells from both pair- and ethanol-fed rats. However, only LPS-stimulated ERK1/2 phosphorylation was sensitive to 10 ng/ml gAcrp. gAcrp also normalized LPS-stimulated DNA binding activity of early growth response-1 with greater sensitivity in Kupffer cells from rats fed chronic ethanol. In conclusion, these results demonstrate that Kupffer cells from ethanol-fed rats are more sensitive to the anti-inflammatory effects of both gAcrp and flAcrp. Suppression of LPS-stimulated ERK1/2 signaling by low concentrations of gAcrp was associated with normalization of TNF-alpha production by Kupffer cells after chronic ethanol exposure.     

Interactions of adiponectin and lipopolysaccharide from Porphyromonas gingivalis on human oral epithelial cells

Background

Periodontitis is an inflammatory disease caused by pathogenic microorganisms, such as Porphyromonas gingivalis, and characterized by the destruction of the periodontium. Obese individuals have an increased risk for periodontitis and show decreased serum levels of adiponectin. This in-vitro study was established to examine whether adiponectin modulates critical effects of lipopolysaccharide (LPS) from P. gingivalis on oral epithelial cells (OECs).

Methodology/Principal Findings

The presence of adiponectin and its receptors in human gingival tissue samples and OECs was analyzed by immunohistochemistry and PCR. Furthermore, OECs were treated with LPS and/or adiponectin for up to 72 h, and the gene expression and protein synthesis of pro- and anti-inflammatory mediators, matrix metalloproteinases (MMPs) and growth factors were analyzed by real-time PCR and ELISA. Additionally, cell proliferation, differentiation and in-vitro wound healing were studied. The nuclear translocation of NFκB was investigated by immunofluorescence. Gingival tissue sections showed a strong synthesis of adiponectin and its receptors in the epithelial layer. In cell cultures, LPS induced a significant up-regulation of interleukin (IL) 1β, IL6, IL8, MMP1 and MMP3. Adiponectin abrogated significantly the stimulatory effects of LPS on these molecules. Similarly, adiponectin inhibited significantly the LPS-induced decrease in cell viability and increase in cell proliferation and differentiation. Adiponectin led to a time-dependent induction of the anti-inflammatory mediators IL10 and heme oxygenase 1, and blocked the LPS-stimulated NFκB nuclear translocation.

Conclusions/Significance

Adiponectin may counteract critical actions of P. gingivalis on oral epithelial cells. Low levels of adiponectin, as observed in obese individuals, may increase the risk for periodontal inflammation and destruction.     

Differential induction of tumor necrosis factor alpha and manganese superoxide dismutase by endotoxin in human monocytes: role of protein tyrosine kinase, mitogen-activated protein kinase, and nuclear factor kappaB

A mutant Escherichia coli lipopolysaccharide (LPS) lacking myristoyl fatty acid markedly stimulates the activity of manganese superoxide dismutase (MnSOD) without inducing tumor necrosis factor alpha (TNFalpha) production by human monocytes (Tian et al., 1998, Am J Physiol 275:C740.), suggesting that induction of MnSOD and TNFalpha by LPS are regulated through different signal transduction pathways. The protein tyrosine kinase (PTK)/mitogen-activated protein kinase (MAPK) pathway plays an important role in the LPS-induced TNFalpha production. In the current study, we determined the effects of PTK inhibitors, genistein and herbimycin A, on the induction of MnSOD and TNFalpha in human monocytes. Genistein (10 microg/ml) and herbimycin A (1 microg/ml) markedly inhibited LPS-induced protein tyrosine phosphorylation, phosphorylation and nuclear translocation of MAPK (p42 ERK, extracellular signal-regulated kinase), and increases in the steady state level of TNFalpha mRNA as well as TNFalpha production. In contrast, at similar concentrations, genistein and herbimycin A had no effect on the LPS-induced activation of nuclear factor kappaB (NFkappaB) and induction of MnSOD (mRNA and enzyme activity) in human monocytes. In addition, inhibition of NFkappaB activation by gliotoxin and pyrrodiline dithiocarbamate, inhibited LPS induction of TNFalpha and MnSOD mRNAs. These results suggest that (1) while PTK and MAPK are essential for the production of TNFalpha, they are not necessary for the induction of MnSOD by LPS, and (2) while activation of NFkappaB alone is insufficient for the induction of TNFalpha mRNA by LPS, it is necessary for the induction of TNFalpha as well as MnSOD mRNAs.     

Lysophosphatidic acid inhibits bacterial endotoxin-induced pro-inflammatory response: potential anti-inflammatory signaling pathways

Previous studies have demonstrated that heterotrimeric guanine nucleotide-binding regulatory (Gi) protein-deficient mice exhibit augmented inflammatory responses to lipopolysaccharide (LPS). These findings suggest that Gi protein agonists will suppress LPS-induced inflammatory gene expression. Lysophosphatidic acid (LPA) activates G protein-coupled receptors leading to Gi protein activation. We hypothesized that LPA will inhibit LPS-induced inflammatory responses through activation of Gi-coupled anti-inflammatory signaling pathways. We examined the anti-inflammatory effect of LPA on LPS responses both in vivo and in vitro in CD-1 mice. The mice were injected intravenously with LPA (10 mg/kg) followed by intraperitoneal injection of LPS (75 mg/kg for survival and 25 mg/kg for other studies). LPA significantly increased the mice survival to endotoxemia (P < 0.05). LPA injection reduced LPS-induced plasma TNF-alpha production (69 +/- 6%, P < 0.05) and myeloperoxidase (MPO) activity in lung (33 +/- 9%, P < 0.05) as compared to vehicle injection. LPS-induced plasma IL-6 was unchanged by LPA. In vitro studies with peritoneal macrophages paralleled results from in vivo studies. LPA (1 and 10 microM) significantly inhibited LPS-induced TNFalpha production (61 +/- 9% and 72 +/- 9%, respectively, P < 0.05) but not IL-6. We further demonstrated that the anti-inflammatory effect of LPA was reversed by ERK 1/2 and phosphatase inhibitors, suggesting that ERK 1/2 pathway and serine/threonine phosphatases are involved. Inhibition of phosphatidylinositol 3 (PI3) kinase signaling pathways also partially reversed the LPA anti-inflammatory response. However, LPA did not alter NFkappaB and peroxisome proliferator-activated receptor gamma (PPARgamma) activation. Inhibitors of PPARgamma did not alter LPA-induced inhibition of LPS signaling. These studies demonstrate that LPA has significant anti-inflammatory activities involving activation of ERK 1/2, serine/threonine phosphatases, and PI3 kinase signaling pathways.     

Adiponectin Inhibits Pro-inflammatory Signaling in Human Macrophages Independent of Interleukin-10

Macrophages participate pivotally in the pathogenesis of many chronic inflammatory diseases including atherosclerosis. Adiponectin, a vasculoprotective molecule with insulin-sensitizing and anti-atherogenic properties, suppresses pro-inflammatory gene expression in macrophages by mechanisms that remain incompletely understood. This study investigated the effects of adiponectin on major pro-inflammatory signaling pathways in human macrophages. We demonstrate that pretreatment of these cells with adiponectin inhibits phosphorylation of nuclear factor κB inhibitor (IκB), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK), induced by either lipopolysaccharide (LPS) or tumor necrosis factor (TNF) α, as well as STAT3 phosphorylation induced by interleukin-6 (IL6). Antagonism of IL10 by either neutralizing antibodies or siRNA-mediated silencing did not abrogate the anti-inflammatory actions of adiponectin, indicating that the ability of adiponectin to render human macrophages tolerant to various pro-inflammatory stimuli does not require this cytokine. A systematic search for adiponectin-inducible genes with established anti-inflammatory properties revealed that adiponectin augmented the expression of A20, suppressor of cytokine signaling (SOCS) 3, B-cell CLL/lymphoma (BCL) 3, TNF receptor-associated factor (TRAF) 1, and TNFAIP3-interacting protein (TNIP) 3. These results suggest that adiponectin triggers a multifaceted response in human macrophages by inducing the expression of various anti-inflammatory proteins that act at different levels in concert to suppress macrophage activation.Adipose tissue, long considered a lipid storage depot, has now gained recognition as an endocrine organ that produces various bioactive molecules with local and systemic functions, collectively known as adipokines (1, 2). Among them, adiponectin has emerged as a key vasculoprotective molecule with insulin-sensitizing, anti-inflammatory, and anti-atherogenic properties (3–5). Numerous (but not all) clinical studies have correlated hypoadiponectinemia with incidence of coronary artery disease, insulin resistance, type 2 diabetes, and hypertension. Experimental studies have demonstrated anti-inflammatory and anti-atherogenic properties of adiponectin by showing that its in vivo overexpression reversed abnormal neointimal thickening in adiponectin-deficient mice, alleviated atherosclerotic lesions in apolipoprotein E-deficient mice, and improved endothelial vasodilator dysfunction and hypertension in obese mice. Cell-based studies demonstrated various potentially anti-atherogenic functions of adiponectin in the major cell types found in atheroma: endothelial cells, smooth muscle cells, and macrophages (3–5).Adiponectin circulates in the plasma at concentrations of 3–30 μg/ml, forming three major oligomeric complexes with distinct biological functions: trimer, hexamer, and high molecular mass form (3–5). A bioactive proteolytic product that includes the adiponectin C1q-like globular domain also exists in plasma, albeit at very low concentrations (6), and in cell culture medium conditioned by THP-1 or U937 cells stimulated with phorbol esters (7).Macrophages contribute critically to the pathogenesis of many chronic inflammatory processes including atherogenesis, and thus comprise key targets for the anti-inflammatory action of adiponectin. Adiponectin inhibits lipopolysaccharide (LPS)²-induced pro-inflammatory gene expression in pig and human macrophages, rat Kupffer cells, and RAW264.7 cells by mechanisms that remain incompletely understood but that involve suppression of LPS-induced nuclear factor κB (NFκB) activation (8–11). Adiponectin induces expression of interleukin-10 (IL10), an immunomodulatory cytokine with potent anti-inflammatory activity, in leukocytes (12, 13). Park et al. (14) recently showed that IL10 generated after treating RAW 264.7 cells with globular adiponectin figures essentially in rendering macrophages tolerant to LPS.We have recently reported that full-length adiponectin inhibits expression of T-lymphocyte-active CXC chemokine receptor 3 (CXCR3) chemokine ligands in human macrophages stimulated by LPS, a process that involves inhibition of interferon (IFN) regulatory factor 3 (IRF3) activation (15). The present study investigated in detail the effects of adiponectin on signaling pathways elicited by the potent pro-inflammatory stimulants LPS, TNFα, and IL6 in human macrophages, and addressed in particular the role of IL10 as a potential mediator of adiponectin function. Our results indicate that adiponectin-induced anti-inflammation in primary human macrophages occurs primarily independently of IL10 and likely involves the concerted action of a group of adiponectin-induced anti-inflammatory molecules that include A20, suppressor of cytokine signaling (SOCS) 3, B-cell CLL/lymphoma (BCL) 3, and TNF receptor-associated factor (TRAF) 1.     

Inhibition by adiponectin of IL-8 production by human macrophages upon coculturing with late apoptotic cells

Adiponectin, an adipocyte-derived hormone, reportedly suppresses the production of TNF-alpha and IL-6 by LPS-stimulated human or porcine macrophages, and the phagocytosis of microbeads by human macrophages. In this study, we used a high molecular weight form of adiponectin purified from human plasma to examine its effects on the phagocytosis of late apoptotic cells by human macrophages and the subsequent IL-8 production. Adiponectin suppressed both the phagocytosis of apoptotic cells and the IL-8 production. In contrast, adiponectin augmented both the phagocytosis of apoptotic cells and the IL-8 production in the presence of LPS. These results suggest that adiponectin is not an anti-inflammatory hormone but rather a dual modulator of innate responses.     

Selective macrophage suppression during sepsis

Polymicrobial sepsis induces suppression of macrophage function as determined by a reduction of pro-inflammatory cytokine production upon re-exposure to lipopolysaccharide (LPS) in vitro. We examined whether macrophages were refractory to only LPS challenge or if they were immunoparalyzed and unable to respond to other stimuli such as lipoteichoic acid (LTA) or zymosan (ZYM). This study evaluated the capacity of peritoneal macrophages to produce pro-inflammatory and anti-inflammatory cytokines as well as chemokines following mild or severe sepsis induced by cecal ligation and puncture (CLP). Peritoneal macrophages were isolated 29 h after CLP and challenged with different stimuli. LPS was a more potent stimulus for cytokine induction than LTA or ZYM in both mild and severe sepsis. In mild sepsis, the macrophage cytokine response to LPS was selective and less refractory than in severe sepsis. While production of IL-6 and KC was reduced, secretion of TNF-alpha and MIP-1alpha was enhanced in those cells isolated from mice with mild sepsis. Production of IL-10 and the IL-1 receptor antagonist , MIP-2, and MCP-1 in response to LPS stimulation was equivalent to the amount produced by naive macrophages. Our results indicate that macrophages are not immunoparalyzed during sepsis and may still be induced to secrete some inflammatory mediators.     

Adiponectin Regulates Vascular Endothelial Growth Factor-C Expression in Macrophages via Syk-ERK Pathway

Adiponectin is exclusively expressed in adipose tissues and exhibits protective effects against cardiovascular and metabolic diseases. It enhances AMP-activated kinase (AMPK) and peroxisome proliferator-activated receptor α (PPARα) signaling in the liver and skeletal muscles, however, its signaling pathways in macrophages remain to be elucidated. Here, we show that adiponectin upregulated the expression of vascular endothelial growth factor (VEGF)-C, and induced phosphorylation of extracellular signal-regulated kinase (ERK) in macrophages. Inhibition of Syk abrogated adiponectin-induced VEGF-C expression and ERK phosphorylation. Furthermore, inhibition of ERK blocked the induction of VEGF-C gene. Inhibition of Syk, but not that of ERK, abrogated adiponectin-induced expression of cyclooxygenase (COX)-2, tissue inhibitor of metalloproteinase (TIMP)-1, and interleukin (IL)-6. These results indicate that adiponectin regulates VEGF-C expression via Syk-ERK pathway in macrophages.     

Royal jelly inhibits the production of proinflammatory cytokines by activated macrophages

In this study, we have examined the anti-inflammatory actions of royal jelly (RJ) at a cytokine level. When supernatants of RJ suspensions were added to a culture of mouse peritoneal macrophages stimulated with lipopolysaccharide and IFN-gamma, the production of proinflammatory cytokines, such as TNF-alpha, IL-6, and IL-1, was efficiently inhibited in a dose-dependent manner without having cytotoxic effects on macrophages. This suggests that RJ contains factor(s) responsible for the suppression of proinflammatory cytokine secretion. We named the factor for honeybees RJ-derived anti-inflammatory factor (HBRJ-AIF), and further investigated the molecular aspects of it. Size fractionation study showed that HBRJ-AIF is composed of substances of low (< 5 kDa) and high (> 30 kDa) molecular weights, with the former being a major component. Chromatographic analysis showed that MRJP3 is one candidate for the HBRJ-AIF with high molecular weights. Thus, our results suggest that RJ has anti-inflammatory actions through inhibiting proinflammatory cytokine production by activated macrophages.     

Human adiponectin binds to bacterial lipopolysaccharide

Adiponectin has anti-inflammatory and anti-atherogenic properties in addition to its acknowledged roles in insulin sensitivity and energy homeostasis. These properties include the suppression of lipopolysaccharide [LPS]-mediated inflammatory events. We demonstrated that both recombinant and native adiponectin directly bind LPS derived from three different bacteria. The interaction occurred at pH 5.0-6.0 and was inhibited by the presence of Ca(2+) and Mg(2+), but enhanced by the sequestration of these cations. Maximal binding occurred at pH 6.0 in the presence of ethylenediaminetetraacetic acid. Lipid A and C1q were not inhibitory, although LPS, heparin, zymosan, and individual sugars all inhibited the reaction. Periodate-mediated deglycosylation of adiponectin, and reduction and alkylation also inhibited binding. Since adiponectin infiltrates into [relatively] acidic sites of inflammation, it may act as a scavenging anti-inflammatory agent in atherosclerosis and vascular damage where LPS [and other pro-inflammatory molecules] are present.     

Regulation of bronchoalveolar macrophage proinflammatory cytokine production by dexamethasone and granulocyte-macrophage colony-stimulating factor after stimulation by Aspergillus conidia or lipopolysaccharide

There is substantial evidence that local production of proinflammatory cytokines are very important in host resistance to aspergillosis. Dexamethasone (DEX) down-regulates production of these cytokines by stimulated bronchoalveolar macrophages (BAM) and constitutes a risk factor for aspergillosis. Granulocyte-macrophage colony-stimulating factor (GM-CSF) antagonizes DEX suppression of antifungal activity by BAM. Here we investigated the possibility that GM-CSF could antagonize DEX down-regulation of interleukin (IL)-1alpha and tumour necrosis factor (TNF)-alpha production by stimulated BAM. Control BAM responded to increasing numbers of conidia of Aspergillus fumigatus with increasing production of IL-1 and TNF. DEX (10(-7)M) significantly suppressed IL-1 and TNF production by BAM+conidia. Although GM-CSF did not enhance IL-1 or TNF production by BAM+conidia, GM-CSF significantly antagonized DEX suppression of IL-1 cytokine production. For comparative purposes, lipopolysaccharide (LPS, 1 microg/ml) was used to stimulate BAM in experiments similar to the above. In contrast to the findings with conidia, GM-CSF enhanced the production of IL-1 (5-fold) and TNF (1.5-fold) by LPS treated BAM. DEX suppression of cytokine production by BAM+LPS was modestly but significantly antagonized by GM-CSF. Moreover, differences between regulation of IL-1 and TNF production by BAM+conidia or LPS and peritoneal macrophages (PM)+conidia or LPS were documented. Finally, the anti-inflammatory cytokine IL-10 was minimally produced by BAM + conidia or LPS, but IL-10 was produced by PM + conidia or LPS. In summary, these data indicate that the risk factor for aspergillosis associated with DEX could be lessened in the pulmonary compartment with GM-CSF. On the other hand, desired effects of DEX could be maintained in other compartments.