Effects of several inhibitors of intracellular signaling on production of cytokines and signal proteins in RAW 264.7 cells cultivated with low dose ammonium

Effects of four inhibitors of NF-kappaB, SAPK/JNK and TLR4 signaling, namely, inhibitor XII, SP600125, CLI-095 and Oxpapc on a macrophage response to low dose ammonium were studied in RAW 264.7 cells. Low dose ammonium induced pro-inflammatory response in cells as judged from enhanced production of TNF-alpha, IF-gamma, and IL-6, and by activation of signal cascades. The increase in production of cytokines, namely TNF, IFN, and IL-6, demonstrated that low-dose ammonium induced a pro-inflammatory cellular response. In addition, an activation of NF-kappaB and SAPK/JNK cascades, as well as enhancement of TLR4 expression was shown. Each of used inhibitors reduced to a variable degree the pro-inflammatory response of RAW 264.7 cells on chemical toxin by decreasing cytokine production. The inhibitor of NF-kappaB cascade, IKK Inhibitor XII, was more effective, and not only prevented the development of pro-inflammatory response induced by ammonium, but also decreased cytokine production below control values. The inhibitor of extra cellular domains of TLR2 and TLR4 (OxPAPC) had almost the same anti-inflammatory effect, and an addition of the inhibitor of JNK cascade (SP600125) to cell culture practically neutralized effect of ammonium ions by decreasing cytokine production to control level. Inhibitory analysis showed that activation of RAW 264.7 cells induced by chemical toxin coincide incompletely with intracellular signaling pathways that were early determined regarding macrophage's response to toxin from gram-negative bacteria. Nevertheless, application of the inhibitors defended RAW 264.7 from toxic effect of the low dose ammonium.     

In vitro and in vivo effects of some inhibitors of signal cascades on cytokines and signal proteins production in raw 264.7 macrophage cells and in mouse lymphocytes

In vitro and in vivo effects of some inhibitors of the activity of signal cascades NF-κB and SAPK/JNK, and the TLR4 receptor on the immune cells activity were studied. To evaluate in vitro effects, the macrophage-like RAW 264.7 cells were cultured with each of the inhibitors, namely IKK inhibitor XII, SP600125, CLI-095, and OxPAPK (the first two are the inhibitors of NF-κB, SAPK/JNK cascades, and the last two compounds are the inhibitors of the TLR4 receptor activity). On the whole, all of the used inhibitors did not induce pro-inflammatory response in RAW 264.7 cells. On the contrary, the inhibitor of SAPK/JNK cascade, and, especially, the inhibitor of NF-κB cascade significantly decreased production of the TNF-α, IL-1, IL-6, IFN-γ, and IL-10 in RAW 264.7 cells. In these cells, the inhibitors substantially decreased “back-ground stress response” of macrophages, differently reducing a production of heat shock proteins, HSP72 and HSP90-α, and diminishing phosphorylation of signal proteins from NF-κB and SAPK/JNK cascades. Results of in vitro experiments suggest that the inhibitor of NF-κB activity was the most effective. It was this inhibitor that was intraperitonealy injected in Balb/C male mice in the in vivo experiments in order to study its effect on the activity of immune cells. Results showed that IKK Inhibitor XII applied in vivo did not induce pro-inflammatory response in mice, but decreased the activity of NF-κB cascade, and lowered HSP90-α expression in mouse splenic lymphocytes. So, among the studied compounds, IKK Inhibitor XII seems to be a very effective inhibitor that may be used to decrease cytokine and stress response in various pathologies.     

The Role of p38 and CK2 Protein Kinases in the Response of RAW 264.7 Macrophages to Lipopolysaccharide

The role of protein kinases p38 and CK2 (casein kinase II) in the response of RAW 264.7 macrophages to the lipopolysaccharide (LPS) from gram-negative bacteria was studied. Using specific p38 and CK2 inhibitors (p38 MAP kinase Inhibitor XI and casein kinase II Inhibitor III, respectively), we investigated the effects of these protein kinases on (i) LPS-induced activation of signaling pathways involving nuclear factor κB (NF-κB), stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), p38, and interferon regulatory factor 3 (IRF3); (ii) expression of Toll-like receptor 4 (TLR4) and inducible heat-shock proteins HSP72 and HSP90; and (iii) production of interleukins IL-1α, IL-1β, IL-6, tumor necrosis factor α, and IL-10. Activation of the proapoptotic signaling in the macrophages was evaluated from the ratio between the active and inactive caspase-3 forms and p53 phosphorylation. Six hours after LPS addition (2.5 μg/ml) to RAW 264.7 cells, activation of the TLR4 signaling pathways was observed that was accompanied by a significant increase in phosphorylation of IκB kinase α/β, NF-κB (at both Ser536 and Ser276), p38, JNK, and IRF3. Other effects of macrophage incubation with LPS were an increase in the contents of TLR4, inducible heat-shock proteins (HSPs), and pro- and anti-inflammatory cytokines, as well as slight activation of the pro-apoptotic signaling in the cells. Using inhibitor analysis, we found that during the early response of macrophages to the LPS, both CK2 and p38 modulate activation of MAP kinase and NF-κB signaling pathways and p65 phosphorylation at Ser276/Ser536 and cause accumulation of HSP72, HSP90 and the LPS-recognizing receptor TLR4. Suppression of the p38 MAP kinase and CK2 activities by specific inhibitors (Inhibitor XI and Inhibitor III, respectively) resulted in the impairment of the macrophage effector function manifested as a decrease in the production of the early-response proinflammatory cytokines and disbalance between the pro- and anti-apoptotic signaling pathways leading presumably to apoptosis development. Taken together, our data indicate the inefficiency of therapeutic application of p38 and CK2 inhibitors during the early stages of inflammatory response.     

Structural characteristics and <Emphasis Type="Italic">in vitro</Emphasis> macrophage activation of acetyl fucoidan from <Emphasis Type="Italic">Cladosiphon okamuranus</Emphasis>

We investigated a structural characteristics of acetyl fucoidan (CAF) isolated from commercially cultured Cladosiphon okamuranus. The CAF-induced macrophage activation and its signaling pathways in murine macrophage cell line, RAW 264.7 were also investigated. From the results of methylation analysis, CAF consisted of α-1→3 linked l-fucosyl residues and substituted sulfate and acetyl groups at C-4 on the main chain. CAF induced production of nitric oxide (NO), tumor necrosis factor-α and interleukin-6 in RAW 264.7 cells. Sulfate and acetyl groups of CAF involved in CAF-induced NO production. Neutralizing anti-Toll-like receptor 4 (TLR4), anti-CD14 and anti-scavenger receptor class A (SRA) but not anti-complement receptor type 3 monoclonal antibodies decreased CAF-induced NO production. The results of immunoblot analysis indicated that CAF activated mitogen-activated protein kinases (MAPKs) such as p38 MAPK, extracellular signal-regulated kinase (ERK)1/2 and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK). SB203580 (p38 MAPK inhibitor) and SP600125 (SAPK/JNK inhibitor), but not U0126 (MAPK/ERK kinase 1/2 inhibitor) decreased CAF-induced NO production. The results suggested that CAF induced macrophage activation through membrane receptors TLR4, CD14 and SRA, and MAPK signaling pathways.     

Immunomodulatory beta-glucan from Lentinus edodes activates mitogen-activated protein kinases and nuclear factor-kappaB in murine RAW 264.7 macrophages

Lentinan, a cell wall β-glucan from the fruiting bodies of Lentinus edodes, is well known to be a biological defense modifier, but the signal transduction pathway(s) induced by Lentinan have not been elucidated. In this study, we extracted Lentinan (LNT-S) by ultrasonication from Lentinus edodes and report that, in murine RAW 264.7 macrophages, LNT-S glucan activated NF-κB p65 and triggered its nuclear translocation as determined by Western blotting. Moreover, LNT-S enhanced NF-κB-luciferase activity in the Dual-Luciferase gene system assay. Its upstream signaling molecules, MAPKs such as ERK1/2 and JNK1/2, were shown to be activated by assessing the level of phosphorylation in a time- and concentration-dependent manner, but its downstream proinflammatory enzyme, inducible NOS, was not observed. The data evaluated using a TNF-α ELISA kit and Griess reagent further demonstrated that no proinflammatory mediators such as TNF-α and NO were produced by LNT-S stimulation in RAW 264.7 cells. In contrast, LPS significantly induced inducible NOS expression and increased NO and TNF-α production, which are associated with activation of the NF-κB p65/p50 heterodimer complex. It is possible that LNT-S did not activate NF-κB p65/p50, and the activation of NF-κB p65 was not sufficient to stimulate cytokine production. These data demonstrate that LNT-S glucan carries out its immunomodulating activity by activating MAPK signaling pathways without secretion of TNF-α and NO.     

The açaí flavonoid velutin is a potent anti-inflammatory agent: blockade of LPS-mediated TNF-α and IL-6 production through inhibiting NF-κB activation and MAPK pathway

Recent studies have shown that some flavonoids are modulators of proinflammatory cytokine production. In this study, velutin, a unique flavone isolated from the pulp of açaí fruit (Euterpe oleracea Mart.), was examined for its effects in reducing lipopolysaccharide-induced proinflammatory cytokine tumor necrosis factor (TNF)-α and interleukin (IL)-6 production in RAW 264.7 peripheral macrophages and mice peritoneal macrophages. Three other structurally similar and well-studied flavones, luteolin, apigenin and chrysoeriol, were included as controls and for comparative purposes. Velutin exhibited the greatest potency among all flavones in reducing TNF-α and IL-6 production. Velutin also showed the strongest inhibitory effect in nuclear factor (NF)-κB activation (as assessed by secreted alkaline phosphatase reporter assay) and exhibited the greatest effects in blocking the degradation of inhibitor of NF-κB as well as in inhibiting mitogen-activated protein kinase p38 and JNK phosphorylation; all of these are important signaling pathways involved in production of TNF-α and IL-6. The present study led to the discovery of a strong anti-inflammatory flavone, velutin. This compound effectively inhibited the expression of proinflammatory cytokines TNF-α and IL-6 in low micromole levels by inhibiting NF-κB activation and p38 and JNK phosphorylation.     

Hydrogen peroxide induces the production of tumor necrosis factor-alpha in RAW 264.7 macrophage cells via activation of p38 and stress-activated protein kinase

The effect of hydrogen peroxide (H(2)O(2)) on production of tumor necrosis factor (TNF)-alpha was examined in RAW 264.7 murine macrophage cells. H(2)O( 2) led to production of TNF-alpha up to 24 h after the treatment, but not nitric oxide in RAW 264.7 cells. H(2)O(2) induced TNF-alpha production in mouse peritoneal macrophages as well as RAW 264.7 cells. The H(2)O(2)induced TNF-alpha production was prevented by inhibitors of p38 and stress-activated protein kinase (SAPK/JNK), and H(2)O( 2) induced the phosphorylation of p38 and SAPK. Further, H(2)O( 2) significantly augmented the AP-1 activity, but not nuclear factor (NF)-kappaB activity in RAW 264.7 cells. A high level of intracellular reactive oxygen radicals (ROS) was detected in H(2)O(2)-exposed RAW 264.7 cells. Ebselen, a cell permeable antioxidant, prevented the H( 2)O(2)-induced TNFalpha production. H(2)O(2) significantly enhanced lipopolysaccharide (LPS)-induced TNF-alpha production. Therefore, H( 2) O(2) was suggested to induce TNF-alpha production in macrophages via activating p38 and SAPK/JNK as oxidative stress-related signal pathways.     

Synergism of Toll-like receptor-induced interleukin-12p70 secretion by monocyte-derived dendritic cells is mediated through p38 MAPK and lowers the threshold of T-helper cell type 1 responses

Toll-like receptors (TLRs) recognise specific molecular signatures of pathogens and trigger antimicrobial defence responses. Thereby, two independent signalling pathways can be distinguished: The inflammatory signalling pathway acting via the adapter molecule MyD88, leading to the activation of nuclear factor-κB (NF-κB) and mitogen activated protein kinases (MAPK) such as SAPK/JNK and p38 MAPK and the interferon (IFN) dependent pathway that signals via TRIF and results in the production of IFN-α/β. Several evolutionarily conserved molecular patterns are expressed by pathogens, leading to the question if concerted targeting of different TLRs may induce exaggerated immune responses by signalling via both TLR pathways. Here we report that monocyte-derived dendritic cells (MoDCs) combine and integrate signals received via the IFN-dependent pathway by engagement of TLR3 (poly I:C) and activation of TRIF with the MyD88-dependent pathway by ligation of TLR2 (PGN), TLR2/TLR6 (zymosan) and TLR5 (flagellin). The generally low IL-12p70 inducers resulted in combination of both pathways in cytokine levels similar to LPS, which acts via TLR4 and induces recruitment of MyD88/Tirap and TRIF/TRAM adapter proteins. The combination of TLR3 (poly I:C) or TLR4 (LPS) engagement with TLR8 (R848) ligation induced synergistic effects on cytokine production with a boost especially in IL-12p70 secretion. SB203580, a specific p38 MAPK inhibitor, completely blocked TLR ligand mediated IL-12p70 secretion, whereby specific inhibitors for SAPK/JNK (SP600125) and NF-κB (PDTC) only repressed partially the IL-12p70 secretion. Enhanced phosphorylation in poly I:C and R848 activated MoDCs revealed the critical contribution of p38 MAPK in synergistically induced IL-12p70 induction. Further investigation of primary and recall CD8⁺ T cell responses to the MUC_12-20 M1.2 peptide LLLLTVLTV and the influenza A virus matrix_58-66 peptide GILGFVFTL proved that synergistically activated MoDCs were superior compared with LPS or R848 alone. The results indicate that dendritic cells process, combine and integrate signals delivered by pathogens to launch effective adaptive immune responses.     

Mechanism of macrophage activation induced by β-glucan produced from Paenibacillus polymyxa JB115

β-Glucans are heterogeneous groups of glucose polymers found in the cell walls of fungi, plants and some bacteria. Our previous report showed that a novel β-1,3/1,6-glucan produced from Paenibacillus (P.) polymyxa JB115 can induce nitric oxide (NO) production in RAW264.7 cells. In the present study, the β-glucan significantly increased luciferase activity in cells transfected with NFκB or AP1, but not STAT1, reporter vector DNA, which contain their binding promoter site. All specific NFκB and MAPKs pathway inhibitors (pyrrolidine dithiocarbamate, AG490, U0126, SB203580 and SP600125) remarkably attenuated NO production induced by the β-glucan. Furthermore, Western blot analysis revealed that the stimulation of Raw264.7 cells by β-glucan induced phosphorylation of IκB and the consequent translocation of NFκB into the nucleus. Meanwhile, phosphorylation of ERK1/2, JNK/SAPK and p38 MAPKs in cytoplasm were also confirmed. All these results indicated that β-glucan from P. polymyxa JB115 activates macrophages through MAPKs and NFκB signaling pathway.     

TNFα and IL-1β are mediated by both TLR4 and Nod1 pathways in the cultured HAPI cells stimulated by LPS

A growing body of evidence recently suggests that glial cell activation plays an important role in several neurodegenerative diseases and neuropathic pain. Microglia in the central nervous system express toll-like receptor 4 (TLR4) that is traditionally accepted as the primary receptor of lipopolysaccharide (LPS). LPS activates TLR4 signaling pathways to induce the production of proinflammatory molecules. In the present studies, we verified the LPS signaling pathways using cultured highly aggressively proliferating immortalized (HAPI) microglial cells. We found that HAPI cells treated with LPS upregulated the expression of TLR4, phospho-JNK (pJNK) and phospho-NF-κB (pNF-κB), TNFα and IL-1β. Silencing TLR4 with siRNA reduced the expression of pJNK, TNFα and IL-1β, but not pNF-κB in the cells. Inhibition of JNK with SP600125 (a JNK inhibitor) decreased the expression of TNFα and IL-1β. Unexpectedly, we found that inhibition of Nod1 with ML130 significantly reduced the expression of pNF-κB. Inhibition of NF-κB also reduced the expression of TNFα and IL-1β. Nod1 ligand, DAP induced the upregulation of pNF-κB which was blocked by Nod1 inhibitor. These data indicate that LPS-induced pJNK is TLR4-dependent, and that pNF-κB is Nod1-dependent in HAPI cells treated with LPS. Either TLR4-JNK or Nod1-NF-κB pathways is involved in the expression of TNFα and IL-1β.     

Analysis of amphotericin B-induced cell signaling with chemical inhibitors of signaling molecules

Although amphotericin B (AmB) is a major polyene antibiotic against invasive fungal infection, administration to patients sometimes causes inflammatory side effects, which limits the usage of the antibiotic. We studied the intracellular signaling that was induced by AmB. p65 (RelA) of nuclear factor‐κB (NF‐κB), a well‐known signaling molecule as an inducer of proinflammatory cytokines, was phosphorylated by AmB in RAW264.7 cells, a monocyte‐like cell line. Among chemical inhibitors of signaling molecules, U‐73122 (phospholipase C (PLC) inhibitor), Gö6976 (protein kinase C (PKC) inhibitor), BAPTA‐AM (calcium chelator), LFM‐A13 (Bruton's tyrosine kinase (Btk)‐specific inhibitor), and PP2 (c‐Src kinase inhibitor) suppressed AmB‐induced phosphorylation of p65 and translocation of p65 into the nucleus. U‐73122 and Gö6976 reduced AmB‐mediated induction of proinflammatory cytokines (tumor necrosis factor (TNF)‐α and interleukin (IL)‐6) in RAW264.7 cells. Furthermore, AmB‐induced activation of NF‐ κ B was observed in toll‐like receptor (TLR) 2‐expressed cells, and the activation of NF‐κB was inhibited by U‐73122, whereas peptidoglycan‐induced NF‐κB activation, which was also dependent on TLR2, was not inhibited by U‐73122. Finally, U‐73122 partially suppressed in vivo production of TNF‐α and IL‐6 induced by AmB administration in BALB/c mice. These results suggested that the signaling from AmB stimulation to proinflammatory cytokine production is mediated by TLR2, Btk, PLC, PKC, c‐Src and NF‐κB. These signaling molecules may become a target for chemotherapy suppressing AmB‐induced proinflammatory cytokine production.     

Role of mitogen-activated protein kinases in the iNOS production and cytokine secretion by Salmonella enterica serovar Typhimurium porins

The expression of inducible nitric oxide synthase (iNOS) is a critical factor in both physiological and pathological functions. The present study examined the role of mitogen-activated protein kinases (MAPKs) in the regulation of iNOS and proinflammatory cytokine production in RAW 264.7 cells in response to Salmonella enterica serovar Typhimurium porins. By use of Western blotting for iNOS detection and enzyme-linked immunosorbent assay (ELISA) for quantization of cytokine secretion, selective pharmacological inhibitors of MAPK pathways were tested for dissecting the molecular mechanisms underlying the mediation of these signaling in porins-stimulated murine macrophages. S. enterica serovar Typhimurium porins activated iNOS expression, NO production and interleukin (IL)-6, IL-8 and tumor necrosis factor-α (TNF-α) release. Treatment of cells with SB203580 and SP600125 (inhibitors of p38 and JNK, respectively) significantly affected porin-stimulated iNOS and NO production. Concomitant decrease in the proinflammatory cytokine secretion was detected. These data confirm the importance of the MAPKs cascade in macrophage activation by bacterial product opening up new strategies for therapy of septic shock.     

Saturated fatty acids activate TLR-mediated proinflammatory signaling pathways

Toll-like receptor 4 (TLR4) and TLR2 were shown to be activated by saturated fatty acids (SFAs) but inhibited by docosahexaenoic acid (DHA). However, one report suggested that SFA-induced TLR activation in cell culture systems is due to contaminants in BSA used for solubilizing fatty acids. This report raised doubt about proinflammatory effects of SFAs. Our studies herein demonstrate that sodium palmitate (C16:0) or laurate (C12:0) without BSA solubilization induced phosphorylation of inhibitor of nuclear factor-κB α, c-Jun N-terminal kinase (JNK), p44/42 mitogen-activated-kinase (ERK), and nuclear factor-κB subunit p65, and TLR target gene expression in THP1 monocytes or RAW264.7 macrophages, respectively, when cultured in low FBS (0.25%) medium. C12:0 induced NFκB activation through TLR2 dimerized with TLR1 or TLR6, and through TLR4. Because BSA was not used in these experiments, contaminants in BSA have no relevance. Unlike in suspension cells (THP-1), BSA-solubilized C16:0 instead of sodium C16:0 is required to induce TLR target gene expression in adherent cells (RAW264.7). C16:0-BSA transactivated TLR2 dimerized with TLR1 or TLR6 and through TLR4 as seen with C12:0. These results and additional studies with the LPS sequester polymixin B and in MyD88(-/-) macrophages indicated that SFA-induced activation of TLR2 or TLR4 is a fatty acid-specific effect, but not due to contaminants in BSA or fatty acid preparations.     

The immunostimulatory effect of indole-6-carboxaldehyde isolated from Sargassum thunbergii (Mertens) Kuntze in RAW 264.7 macrophages

ABSTRACT

Indole-6-carboxaldehyde (I6CA), an indole derivative isolated from the marine brown algae Sargassum thunbergii, is known to have several beneficial effects, but no studies on immune regulation have been conducted. In this study, the immunomodulatory properties of I6CA on murine RAW 264.7 monocyte/macrophage cells were evaluated. As the concentration of I6CA increased, the morphology of RAW 264.7 cells changed to a typical active macrophage shape, and the phagocytic activity increased significantly. I6CA effectively enhanced the production and secretion of immunomodulatory mediators and cytokines due to increased expression of their respective genes. Additionally, I6CA markedly stimulated the expression of Toll-like receptor 4 (TLR4) and its adapter molecule, myeloid differentiation factor 88 (Myd88), and increased TLR4 complexed with Myd88. Furthermore, I6CA promoted the nuclear translocation of nuclear factor-kappa B (NF-κB) by increasing the degradation of the inhibitor of NF-κB-α. Meanwhile, similar trends were also found in lipopolysaccharide-treated cells as a positive control. Furthermore, molecular docking simulation showed that I6CA interacted with TLR4-myeloid differentiation 2 complex. Taken together, the results support the concept that I6CA may increase the activity of the TLR4/NF-κB signaling pathway in order to enhance the immunomodulatory activity of RAW 264.7 cells.