Phagocytic Activity Of RAW264 Research Articles

Preparation Optimization and Immunological Activity Studies of Portulaca oleracea L. Polysaccharides Liposomes.

This study combines traditional Chinese medicine polysaccharides with nanomaterials to enhance drug bioavailability and immunological activity. The study of polysaccharide preparation, structure identification, pharmacological activity, and mechanism of action is deepening, but the research combined with the new drug delivery system is relatively weak, so the application of polysaccharides is still facing great limitations. In order to prolong the action time of polysaccharides and improve their bioavailability, liposome has become the most promising delivery carrier. The purpose of this study was to optimize the preparation process of Portulaca oleracea L. polysaccharides liposomes (POL-PL) and evaluate the immunoactivity in vitro. POL-PL was prepared by reverse evaporation, and the preparation process was optimized using the response surface methodology. The characteristic analysis of POL-PL was detected by the indicators including morphology, particle size, zeta potential, encapsulation efficiency, release, and stability. The effects of POL-PL on the proliferation and immunological activity of mouse spleen lymphocytes and RAW264.7 cells were evaluated in vitro. POL-PL is highly homogeneous in morphology and particle size, and its sustained release improves the bioavailability of Portulaca oleracea L. polysaccharides (POL-P). Moreover, POL-PL treatment significantly enhanced the proliferation and phagocytic activity of RAW264.7 cells and increased the secretion of IL-6, TNF-α, IL-1β, and NO. This study suggested that POL-PL were prepared successfully by reverse evaporation method, and POL-PL had immunoenhancing activity in vitro. The results provided a theoretical basis for further application of POL-PL.

Immune Enhancement Effects of Neutral Lipids, Glycolipids, Phospholipids from Halocynthia aurantium Tunic on RAW264.7 Macrophages.

Fractionated lipids of Halocynthia aurantium (Pyuridae) have been demonstrated to possess anti-inflammatory properties. However, their modulatory properties have not been reported yet. Thus, the objective of this study was to determine immune enhancing effects of fractionated lipids from H. aurantium tunic on macrophage cells. The tunic of H. aurantium was used to isolate total lipids, which were then subsequently separated into neutral lipids, glycolipids, and phospholipids. RAW264.7 cells were stimulated with different concentrations (0.5, 1.0, 2.0, and 4.0%) of each fractionated lipid. Cytotoxicity, production of NO, expression levels of immune-associated genes, and signaling pathways were then determined. Neutral lipids and glycolipids significantly stimulated NO and PGE2 production and expression levels of IL-1β, IL-6, TNF-α, and COX-2 in a dose-dependent manner, while phospholipids ineffectively induced NO production and mRNA expression. Furthermore, it was found that both neutral lipids and glycolipids increased NF-κB p-65, p38, ERK1/2, and JNK phosphorylation, suggesting that these lipids might enhance immunity by activating NF-κB and MAPK signaling pathways. In addition, H. aurantium lipids-induced TNF-α expression was decreased by blocking MAPK or NF-κB signaling pathways. Phagocytic activity of RAW 264.7 cells was also significantly enhanced by neutral lipids and glycolipids. These results suggest that neutral lipids and glycolipids from H. aurantium tunic have potential as immune-enhancing materials.

Discrimination of Radix Astragali from Different Growth Patterns, Origins, Species, and Growth Years by an H1-NMR Spectrogram of Polysaccharide Analysis Combined with Chemical Pattern Recognition and Determination of Its Polysaccharide Content and Immunological Activity.

The fraud phenomenon is currently widespread in the traditional Chinese medicine Radix Astragali (RA) market, especially where high-quality RA is substituted with low-quality RA. In this case, focused on polysaccharides from RA, the classification models were established for discrimination of RA from different growth patterns, origins, species, and growth years. 1H Nuclear Magnetic Resonance (H1-NMR) was used to establish the spectroscopy of polysaccharides from RA, which were used to distinguish RA via chemical pattern recognition methods. Specifically, orthogonal partial least squares discriminant analysis (OPLS-DA) and linear discriminant analysis (LDA) were used to successfully establish the classification models for RA from different growth patterns, origins, species, and growth years. The satisfactory parameters and high accuracy of internal and external verification of each model exhibited the reliable and good prediction ability of the developed models. In addition, the polysaccharide content and immunological activity were also tested, which was evaluated by the phagocytic activity of RAW 264.7. And the result showed that growth patterns and origins significantly affected the quality of RA. However, there was no significant difference in the aspects of origins and growth years. Accordingly, the developed strategy combined with chemical information, biological activity, and multivariate statistical method can provide new insight for the quality evaluation of traditional Chinese medicine.

Identification and Molecular Mechanism of Novel Immunomodulatory Peptides from Gelatin Hydrolysates: Molecular Docking, Dynamic Simulation, and Cell Experiments.

The purpose of this study was to identify donkey-hide gelatin-derived immunomodulatory peptides targeting Toll-like receptor 4-myeloid differentiation 2 (TLR4-MD2) and elucidate their binding modes using physicochemical property prediction, molecular docking, molecular dynamics simulations, and in vitro cell experiments. After hydrolyzing gelatin, 519 peptides were identified by liquid chromatography-tandem mass spectrometry. Peptides VQLSGEEK and GFSGLDGAKG bound to TLR4-MD2 with high binding affinity. In TLR4-MD2, Arg90, Ser118, Phe126, Tyr131, and Arg264 were key residues involved in the binding of these peptides. The RMSD and Rg values demonstrated that VQLSGEEK-TLR4-MD2 and GFSGLDGAKG-TLR4-MD2 complexes had stable and compact conformations. VQLSGEEK and GFSGLDGAKG were found to increase the cell viability and phagocytic activity of RAW264.7 macrophages; significantly promote the production of cytokines TNF-α, IL-1β, and IL-6 in cells; and inhibit the overproduction of nitric oxide (NO) and cytokines in lipopolysaccharide (LPS)-induced RAW264.7 cells. Our results provided preliminary evidence that VQLSGEEK and GFSGLDGAKG could function as two-way immunomodulatory peptides with immunostimulatory and anti-inflammatory activities.

Structural characterization of a soluble polysaccharide SSPS1 from soy whey and its immunoregulatory activity in macrophages

A soluble soybean polysaccharide SSPS1 with a molecular weight of 2737 kDa was extracted and purified from soy whey. SSPS1 was composed of glucose (97.3 %) and a small amount of mannose (2.7 %). Structural analysis results suggested that SSPS1 had a → 6)-α-d-Glcp-(1 → glucan structure, with a trace amount of α-d-Glcp-(1 → connected to the main chain via O-3. In vitro immunological experiments suggested that SSPS1 enhanced the growth rate and phagocytic activity of RAW 264.7 macrophages. In addition, SSPS1 stimulated the secretion of cytokines (TNF-α, INF-β, IL-6 and IL-1β) as well as nitric oxide (NO) production through upregulating the expression of the related genes and proteins in RAW 264.7 cells. This study provided a new method for efficient utilization of soy whey, and the results indicate that SSPS1 extracted from soy whey could be used as a novel immunomodulator.

The Immunomodulatory Effects of Active Ingredients From Nigella sativa in RAW264.7 Cells Through NF-κB/MAPK Signaling Pathways.

Nigella sativa is a valuable herb for its functional compositions in both food and medication. N. sativa seeds can enhance immunity, anti-inflammation and analgesia and hypoglycemia, but most of the related researches are related to volatile oil and extracts, and the activity and mechanism of compounds is not clear. In this study, Ethyl-α-D-galactopyranoside (EG), Methyl-α-D-glucoside (MG), 3-O-[β-D-xylopyranose-(1 → 3)-α-L-rhamnose-(1 → 2)-α-L-arabinose]-28-O-[α-L-rhamnose-(1 → 4)-β-D-glucopyranose-L-(1 → 6)-β-D-glucopyranose]-hederagenin (HXRARG) and 3-O-[β-D-xylopyranose-(1 → 3)-α-L-rhamnose-(1 → 2)-α-L-arabinose]-hederagenin (HXRA) were isolated and identified from N. sativa seeds. In addition, four compounds could activate NF-κB pathway by promoting the expression of phosphorylation of P65 and IκBα, promoting the phosphorylation of JNK, Erk and P38 to activate MAPK signaling pathway, enhancing the proliferation and phagocytic activity of RAW264.7 cells, and promoting the release of NO, TNF-α and IL-6 on RAW264.7 cell in vitro. The results showed that N. sativa can be used as dietary supplement to enhance immune.

Immunostimulatory activity of stem bark of Kalopanax pictus in RAW 264.7 macrophage

The purpose of this study was to examine the potential immune-enhancing effects of hot water extracts from Kalopanax pictus (KPW) stem barks on mouse macrophage RAW 264.7 cells. Cytotoxicity and nitric oxide (NO) production were measured after KPW treatment via MTT and Griess assays, respectively. The levels of immune-related proteins such as iNOS and COX-2 were determined by Western blot analysis, and cytokines related to immune responses and phagocytic activity were examined by ELISA. KPW significantly increased NO production in a concentration-dependent manner without any cytotoxicity. In addition, the levels of certain enzymes (iNOS and COX-2) and cytokines (TNF-α, IL-1β, IL-6, and IL-12) were also significantly upregulated. Furthermore, the phagocytic activity of RAW 264.7 cells was significantly enhanced by KPW treatment. Taken together, these results suggest that KPW have a potential as an immunostimulatory agent.

Immune-enhancing screening of fourteen plants on murine macrophage RAW 264.7 cells

Purpose: To investigate the potential immune-enhancing effects of fourteen natural plant extracts on mouse macrophage RAW 264.7 cells.Methods: Fourteen plant extracts from 7 different plants were tested on RAW 264.7 cells to determine their immunostimulant activities. Methylthiazolydiphenyltetrazolium bromide (MTT) and Griess assays were performed to evaluate cell viability and nitric oxide (NO) production, respectively. Then, immune related proteins were measured by western blot analysis, while cytokines and phagocytic activity were determined by enzyme-linked immunosorbent assay (ELISA) method.Results: Among the 14 plant extracts, the hot water extract of Agastache rugose was selected based on the screening results on NO production. The hot water extract of A. rugose significantly increased NO production in a concentration-dependent manner without any cytotoxicity. In addition, the expression levels of proteins (iNOS and COX-2) and cytokines (TNF-α, IL-1β, IL-6 and IL-12) closely related to immune reaction were also significantly upregulated. Furthermore, phagocytic activity of RAW 264.7 cells significantly increased following treatment with A. rugosa.Conclusion: The hot water extract of A. rugosa exhibits significant immune-stimulant activity. Therefore, A. rugosa can be used as a natural resource for immune enhancement or dietary supplement.Keywords: Immune enhancing activity, Macrophage polarization, Natural plant extracts, Agastache rugosa, RAW 264.7

Chlorogenic acid protects against aluminum toxicity via MAPK/Akt signaling pathway in murine RAW264.7 macrophages

Aluminum (Al), which may bring about damage to the macrophages, has been implicated in the development of immunological diseases. It has been reported that chlorogenic acid (CGA, 5‑caffeoylquinic acid, chemical formula: C16H18O9) is a natural antioxidant and chelating agent with the capacity against Al (III)-induced biotoxicity. The present study was carried out to investigate whether CGA could reduce AlCl3-induced cellular damage in RAW264.7 cells. After treatment with AlCl3, the inhibition rate of viability and phagocytic activity of RAW264.7 cells was 54.5% and 27.6%, respectively. Administration of CGA significantly improved the integrity and phagocytic activity, and attenuated the accumulation of intracellular Al(III) level and oxidative stress in Al(III)-treated cells. Furthermore, CGA significantly inhibited Al(III)-induced increase of phospho-Jun N-terminal kinase (p-JNK), a pro-apoptotic Bcl-2 family protein (Bad), cytochrome c and decrease of extracellular regulated protein kinases (ERK1/2), protein kinase B (Akt) protein expressions. These results showed that CGA has a protective effect against Al(III)-induced cytotoxicity through mitogen-activated protein kinase (MAPK)/Akt-mediated caspase pathways in RAW264.7 cells.

Immune-enhancing activity of C. militaris fermented with Pediococcus pentosaceus (GRC-ON89A) in CY-induced immunosuppressed model

BackgroundCordyceps militaris (C. militaris) is reported to exert various immune-activities. To enhance its activity, we fermented C.militaris with Pediococcus pentosaceus ON89A (GRC-ON89A). In this study, we investigated the immune-enhancing activity GRC-ON89A, using immunosuppressed model.MethodsImmunosuppression was induced by intraperitoneal injection of cyclophosphamide (CY). Each group was orally administered distilled water, GRC-ON89A or GRC, respectively. The phagocytic activities against IgG -opsonized FITC particles were measured using phagocytosis assay kit. The contents β-glucan, cordycepin and SCFA were measured using β-glucan kit, liquid chromatography-mass spectrometry analysis and Gas chromatography-mass spectrometry analysis, respectively.ResultsAmong GRC fermented with different probiotic strains (Pediococcus pentossaceus ON89A, Lactobacillus pentosus SC64, Weissella cibaria Sal.Cla22), GRC-ON89A induced the highest elevation of nitric oxide production and enhanced phagocytic activity of RAW 264.7 cells. In primary cultured murine macrophages from normal and CY-treated mice, GRC-ON89A increased phagocytic activity, compared to that in control cells. GRC-ON89A also significantly induced the mRNA expression of TNF-α and IL-10 and the levels of phosphorylated Lyn, Syk and MAPK. The contents of β-glucan, cordycepin and SCFA in GRC significantly increased after ON89A fermentation, compared to those in unfermented GRC.ConclusionThese results indicate that GRC-ON89A exerted the enhanced immunostimulatory activity and contained more nutritional components, compared to unfermented GRC. Our results suggested that GRC-ON89A may be applied as an agent for immune boosting therapy in immune suppressed patients.

Immune-enhancing activity of phosvitin by stimulating the production of pro-inflammatory mediator

Egg yolk phosvitin is one of the most phosphorylated proteins in nature, and the extraordinarily high concentration of phosphate groups in its structure provides a strong metal-binding ability. Phosvitin is known to possess various functional activities, including metal-chelating, antioxidant, emulsifying, antimicrobial, and cytotoxic activities. However, little is known about the immune-enhancing activity of phosvitin. The objective of this study was to evaluate the immune-enhancing activity of phosvitin in murine RAW 264.7 macrophages. Griess reagents and quantitative real-time PCR were used to determine the effect of phosvitin (at 12.5, 25, 50, and 100 μg/mL) on the levels of pro-inflammatory mediators NO and inducible nitric oxide synthase (iNOS), cytokines TNF-α and IL-1β in RAW 264.7 macrophages. The effect of phosvitin on the phagocytic activity of RAW 264.7 macrophages was also measured using the Neutral-Red Uptake method. Lipopolysaccharides was used as a positive control. Phosvitin significantly (P < 0.05) increased the production of NO in RAW 264.7 macrophages in a dose-dependent manner, but did not show any cytotoxicity. The amounts of NO produced were 3.47, 7.12, 10.23, and 14.57 μM in 12.5 to 100 μg/mL range of phosvitin (control: 0.46 μM). Compared with the untreated group, phosvitin treatment at a 100 μg/mL level increased the production of NO by 31.67 times. Phosvitin also significantly increased the mRNA expression of the RAW 264.7 macrophages: 100 μg/mL of phosvitin treatment increased the expression of mRNA for iNOS, TNF-α, and IL-1β by 46.25, 9.09, and 85.18 times of the control, respectively. The phagocytic activity of RAW 264.7 macrophages was also increased significantly by phosvitin treatment. These results demonstrated that phosvitin dramatically improved the immune functions RAW 264.7 macrophages by enhancing the production of immune mediators and increasing phagocytic activity. Therefore, phosvitin has a potential to be used as an immune-enhancing agent by food or nutraceutical industries.

Immunobiological Activity of Synthetically Prepared Immunodominant Galactomannosides Structurally Mimicking Aspergillus Galactomannan.

The study is oriented at the in vitro evaluation of the immunobiological activity and efficacy of synthetically prepared isomeric pentasaccharides representing fragments of Aspergillus fumigatus cell-wall galactomannan and containing β-(1→5)-linked tetragalactofuranoside chain attached to O-6 (GM-1) or O-3 (GM-2) of a spacer-armed mannopyranoside residue. These compounds were studied as biotinylated conjugates which both demonstrated immunomodulatory activities on the RAW 264.7 cell line murine macrophages as in vitro innate immunity cell model. Immunobiological studies revealed time- and concentration-dependent efficient immunomodulation. The proliferation of RAW 264.7 macrophages was induced at higher concentration (100 µg/mL) of studied glycoconjugates and longer exposure (48 h), with more pronounced efficacy for GM-1. The increase of proliferation followed the previous increase of IL-2 production. The cytokine profile of the macrophages treated with the glycoconjugates was predominantly pro-inflammatory Th1 type with significant increase of TNFα, IL-6, and IL-12 release for both glycoconjugates. The RAW 264.7 macrophages production of free radicals was not significantly affected by glycoconjugates stimulation. The phagocytic activity of RAW 264.7 cells was reduced following GM-1 treatment and was significantly increased after 24 h stimulation with GM-2, contrary to 48 h stimulation. Moreover, the synthetically prepared galactomannoside derivatives have been evaluated as efficient serodiagnostic antigens recognized by specific Ig isotypes, and significant presence of specific IgM antibodies in serum of patients suffering from vulvovaginitis was observed.

Water extract of Glycyrrhiza uralensis Fisch. enhanced the activation of RAW264.7 cells through TLR4 signaling pathway

Objective To investigate the effects of water extract of Glycyrrhiza uralensis Fisch. (GUWE) on the activation of RAW264.7 cells and the possible mechanism. Methods RAW264.7 cells were treated with GUWE containing different concentrations of polysaccharide (10, 50, 100, 500 μg/ml). Viability of these cells was analyzed by MTT assay. Phagocytic activity and surface molecules expressed on these cells were detected by flow cytometry. Levels of cytokines were analyzed by ELISA. Western blot assay was performed to analyze the activation of key molecules in TLR4 signaling pathway. Results GUWE at the concentration of 500 μg/ml significantly decreased the viability of RAW264.7 cells, but significantly increased the viability of RAW264.7 cells at concentrations of 50 μg/ml and 100 μg/ml. GUWE significantly enhanced the phagocytic activity of RAW264.7 cells as well as the expression of cytokines and costimulatory molecules in a dose-dependent manner. Further analysis indicated that the activation of RAW264.7 cells induced by GUWE was suppressed by TLR4 inhibitor. Moreover, GUWE enhanced the phosphorylation of NF-κB p65 and TLR4 downstream mitogen-activated protein kinases (MAPKs) including p38, extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK). Conclusion This study indicates that GUWE promotes the activation of RAW264.7 cells through TLR4 signaling pathway. Key words: Water extract of Glycyrrhiza uralensis Fisch.; RAW264.7 cell; Costimulatory molecule; Cytokine; TLR4

In vitro and in vivo assessment of anti-inflammatory and immunomodulatory activities of Xylaria nigripes mycelium

Xylaria nigripes (XN) is used to treat insomnia, trauma and inflammatory diseases. This study demonstrated the anti-inflammatory and immunomodulatory activities of XN. Submerged fermentation was applied for production of XN mycelium. For in vitro studies on the anti-inflammation activity, hot water and 70% ethanol extracts of XN mycelia were prepared. Lipopolysaccharide-induced RAW264.7 cells served as the cell culture model to evaluate the effects of XN extracts against disease-related inflammation. The immunomodulatory effects were examined by oral administration of XN mycelium in BALB/c mice. Both extracts effectively reduced the concentration of NO, TNF-α and IL-6 in culture medium, and Cox-2 enzyme activity. The phagocytic activity of RAW264.7cells was enhanced upon treatment with XN extracts for 24h. XN extract exhibited increased anti-inflammation effects, phagocytic activity, and secretion of anti-inflammatory cytokine IL-10. The XN mycelium exhibited immunomodulatory activities by enhancing splenocytes proliferation, cytokine inductions, and the cytotoxicity of splenic natural killer cells.

Extraction, Characterization and Immunological Activity of Polysaccharides from Rhizoma gastrodiae.

A response surface and Box-Behnken design approach was applied to augment polysaccharide extraction from the residue of Rhizoma gastrodiae. Statistical analysis revealed that the linear and quadratic terms for three variables during extraction exhibited obvious effects on extraction yield. The optimum conditions were determined to be a liquid-to-solid ratio of 54 mL/g, an extraction temperature of 74 °C, an extraction time of 66 min, and three extractions. These conditions resulted in a maximum Rhizoma gastrodiae polysaccharide (RGP) extraction yield of 6.11% ± 0.13%. Two homogeneous polysaccharides (RGP-1a and RGP-1b) were obtained using DEAE cellulose-52 and Sephadex G-100 columns. The preliminary characterization of RGP-1a and RGP-1b was performed using HPLC-RID, HPGPC, and FTIR. Tests of the immunological activity in vitro showed that the two polysaccharides could significantly stimulate macrophages to release NO and enhance phagocytosis in a dose-dependent manner. In particular, RGP-1b (200 μg/mL) and LPS (2 μg/mL) had almost the same influence on the NO production and phagocytic activity of RAW 264.7 macrophages (p > 0.05). All the data obtained indicate that RGP-1a and RGP-1b have the potential to be developed as a health food.

Effects of Glycated Whey Protein Concentrate on Pro-inflammatory Cytokine Expression and Phagocytic Activity in RAW264.7 Macrophages

The aim of this study was to determine the stimulatory effects of Maillard reaction, a non-enzymatic browning reaction on the expression of pro-inflammatory cytokines and phagocytic activity induced by whey protein concentrate (WPC). Glycated WPC (G-WPC) was prepared by a reaction between WPC and the lactose it contained. The fluorescence intensity of G-WPC dramatically increased after one day, and high molecular weight complexes formed via the Maillard reaction were also observed in the sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles. G-WPC demonstrated immunomodulatory effects, including stimulation of increased nitric oxide production and cytokine expressions (i.e., tumor necrosis factor-α, interleukin (IL)-1β, and IL-6), compared to WPC. Furthermore, the phagocytic activity of RAW264.7 cells was significantly increased upon treatment with G-WPC, compared to WPC. Therefore, we suggest that G-WPC can be utilized as an improved dietary source for providing immune modulating activity.

Polysaccharides from Umbilicaria esculenta cultivated in Huangshan Mountain and immunomodulatory activity

Umbilicaria esculenta cultivated in Huangshan Mountain (HSSE) is precious edible and medicinal lichen. In this study, four polysaccharide fractions designated as UEP1, UEP2, UEP3, and UEP4 were isolated from HSSE with water extraction at different temperature. The physico-chemical properties and immunomodulatory activities of polysaccharide fractions were investigated. The results indicated that UEP1, UEP2, UEP3 and UEP4 were acid polysaccharide with 0.50%, 0.62%, 0.63%, and 0.83% of uronic acid contents, respectively. Four polysaccharide fractions were mainly composed of glucose, galactose and mannose with different molar ratio. In the in vitro immunomodulatory assay, all the polysaccharide fractions (20–500μg/mL) could increase the NO production and phagocytic activity of RAW 264.7 cells in a dose-dependent manner. This work demonstrated that the polysaccharides from HSSE could be used as potential biological response modifier.

CD83 expression induced by CpG-DNA stimulation in a macrophage cell line RAW 264.7

CpG-DNA has various immunomodulatory effects in dendritic cells, B cells, and macrophages. While induction of cytokines by CpG-DNA has been well documented in macrophages, the expression of costimulatory molecules in CpG-DNA treated macrophages has not yet been defined. Therefore, we investigated the effects of CpG-DNA on the expression of costimulatory molecules in RAW 264.7 cells. The surface expression of CD80 was slightly increased and CD83 expression was significantly increased in response to CpG-DNA. However, the expression of CD86 and MHC class II was not changed. As expression of CD83 mRNA was also increased by CpG-DNA, CD83 expression is regulated at a transcriptional level. To understand the contribution of signaling pathways to CD83 induction, we used pathway specific inhibitors. The NF-κB inhibitor significantly reduced surface expression of CD83 as well as phagocytic activity of RAW 264.7 cells. Therefore, CD83 expression may contribute to the immunostimulatory effects of CpG-DNA in macrophage cells. [BMB Reports 2013; 46(9): 448-453]

The uraemic toxin phenylacetic acid impairs macrophage function

Nitric oxide (NO) is known to be an important mediator of macrophage cytotoxicity. NO in macrophages is generated via the inducible nitric oxide synthases (iNOS). Macrophage dysfunction is an important contributory factor for the increased incidence of infections in uraemia. Recently, we identified phenylacetic acid (PAA) as a novel uraemic toxin in patients on regular haemodialysis. PAA inhibits iNOS expression. In the present study, we investigated the impact of PAA on macrophage function. RAW 264.7 cells were stimulated by LPS/ IFN-gamma in the absence and presence of PAA. iNOS mRNA was determined by real-time PCR, iNOS protein was examined by western blotting and the NO degradation product, nitrite, by Griess assay. Macrophage phagocytosis was assessed by FACS and fluorescence microscopy. Further we quantified the cytotoxicity against intracellular bacteria (Salmonella typhimurium) by a macrophage-killing assay. ELISA and Bioplex protein array system was used for the investigation of iNOS second messenger pathways (NF-kappaB, ERK1/2, JNK and p38MAPK). iNOS mRNA half-lifetime in the presence or absence of PAA was determined by real-time PCR. PAA significantly inhibits iNOS mRNA induction in RAW 264.7 cells by LPS/IFN-gamma [6 h: LPS/IFN-gamma-stimulation: 100%; LPS/IFN-gamma-stimulation/PAA (1 mM): 68 +/- 7%] at concentrations comparable to those of patients on chronic haemodialysis. iNOS protein expression and nitrite formation in RAW 264.7 cells were significantly inhibited by PAA. iNOS mRNA half-lifetime was not affected by PAA. The phagocytic activity of RAW 264.7 was not significantly affected by PAA, whereas the cytotoxicity against intracellular bacteria was significantly reduced. Analysis of the iNOS signal transduction pathways provided evidence that activation of the mitogen-activated kinases ERK1/2 and JNK is significantly blocked by PAA, whereas activation of p38MAPK is unaffected. The NF-kappaB pathway was not affected by PAA. The present findings show that the uraemic toxin PAA has inhibitory effects on macrophage-killing function, which are mediated by inhibitory effects on transcriptional iNOS regulation. iNOS inhibition by PAA might affect immunoregulatory processes and could play a role in aggravation of immunodeficiency of patients with end-stage renal disease.