TNF-alpha Production In Macrophages Research Articles

Atractylenolide I and atractylenolide III inhibit Lipopolysaccharide‐induced TNF‐α and NO production in macrophages

In order to clarify the mechanism involved in the antiinflammatory activity of atractylenolide I and atractylenolide III from the rhizomes of Atractylodes macrocephala Koidz, their effects on tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) production in peritoneal macrophages were examined. Atractylenolide I and atractylenolide III decreased the TNF-alpha level in LPS-stimulated peritoneal macrophages in a dose-dependent manner, their IC(50) values were 23.1 microm and 56.3 microm, respectively. RT-PCR analysis indicated that they inhibited TNF-alpha mRNA expression. Furthermore, they inhibited NO production in LPS-activated peritoneal macrophages, the IC(50) value of atractylenolide I was 41.0 microm, and the inhibition ratio of 100 microm of atractylenolide III was 45.1% +/- 6.2%. The activity analysis of inducible nitric oxide synthase (iNOS) indicated that they could inhibit the activity of iNOS, their IC(50) values were 67.3 microm and 76.1 microm, respectively. Western blot analysis showed that atractylenolide I and atractylenolide III attenuated LPS-induced synthesis of iNOS protein in the macrophages, in parallel. These results imply that the antiinflammatory mechanism of atractylenolide I and atractylenolide III may be explained at least in part, by the inhibition of TNF-alpha and NO production. Atractylenolide I showed more potent inhibition than atractylenolide III in the production of TNF-alpha and NO in LPS-activated peritoneal macrophages. So, atractylenolide I could be a candidate for the development of new drugs to treat inflammatory diseases accompanied by the overproduction of TNF-alpha and NO.

Dysregulation of interleukin‐10–dependent gene expression in rheumatoid arthritis synovial macrophages

The inflammatory cytokines tumor necrosis factor alpha (TNFalpha) and interleukin-1 (IL-1) are produced by activated macrophages, are key mediators of pathogenesis, and are validated therapeutic targets in rheumatoid arthritis (RA) and seronegative spondylarthritis (SpA). IL-10 is a potent antiinflammatory cytokine that suppresses macrophage TNFalpha and IL-1 production, yet is not effective in suppressing inflammatory arthritis. To gain insight into IL-10 responses in inflammatory arthritis, we used microarray analysis to determine the patterns of IL-10-inducible gene expression in freshly isolated RA and seronegative SpA synovial macrophages. Macrophages from the synovial fluid of 5 patients with RA and 3 with seronegative SpA (2 with psoriatic arthritis and 1 with ankylosing spondylitis) were isolated by positive selection and stimulated ex vivo with IL-10 or interferon-gamma (IFNgamma). Gene expression was analyzed using Affymetrix microarrays and protocols. Real-time polymerase chain reaction was used to confirm changes in gene expression. The number of genes induced by IL-10 in arthritic macrophages was markedly smaller than that induced in control macrophages, and the strength of induction was lower in arthritic macrophages for most genes. The residual response of arthritic macrophages to IL-10 stimulation was qualitatively altered, such that IL-10 preferentially increased expression of IFNgamma-inducible genes. In contrast, arthritic macrophages expressed many IFNgamma-inducible genes prior to stimulation, and their response to IFNgamma remained mostly intact. These results demonstrate that IL-10 responses are dysregulated in RA synovial macrophages. An altered biologic response to IL-10, with attenuation of its antiinflammatory function and a concomitant retention of IFNgamma-like activating functions, provides a basis for the lack of efficacy of IL-10 in suppressing inflammatory arthritis.

Ca2+- and Protein Kinase C-dependent Signaling Pathway for Nuclear Factor-κB Activation, Inducible Nitric-oxide Synthase Expression, and Tumor Necrosis Factor-α Production in Lipopolysaccharide-stimulated Rat Peritoneal Macrophages

Lipopolysaccharide (LPS)-activated macrophages are pivotal in innate immunity. With LPS treatment, extracellular signals are transduced into macrophages via Toll-like receptor 4 and induce inflammatory mediator production by activating signaling pathways, including the nuclear factor-kappaB (NF-kappaB) pathway and the mitogen-activated protein kinase (MAPK) pathway. However, the mechanisms by which the intracellular free Ca2+ concentration ([Ca2+]i) increases and protein kinase C (PKC) is activated remain unclear. Therefore, we investigated the signaling pathway for Ca2+- and PKC-dependent NF-kappaB activation, inducible nitric-oxide synthase expression, and tumor necrosis factor-alpha (TNF-alpha) production in LPS-stimulated rat peritoneal macrophages. The results demonstrated that the LPS-induced transient [Ca2+]i increase is due to Ca2+ release and influx. Extracellular and intracellular Ca2+ chelators inhibited phosphorylation of PKCalpha and PKCbeta. A PKCbeta-specific and a general PKC inhibitor blunted phosphorylation of serine in mitogen-activated/extracellular signal-regulated kinase kinase kinase (MEKK) 1. Moreover, a MEKK inhibitor reduced activation of inhibitorykappaB kinase and NF-kappaB. Upstream of the [Ca2+]i increase, a protein-tyrosine kinase inhibitor reduced phosphorylation of phospholipase C (PLC) gamma. Furthermore, a PLC inhibitor eliminated the transient [Ca2+]i increase and decreased the amount of activated PKC. Therefore, these results revealed the following roles of Ca2+ and PKC in the signaling pathway for NF-kappaB activation in LPS-stimulated macrophages. After LPS treatment, protein-tyrosine kinase mediates PLCgamma1/2 phosphorylation, which is followed by a [Ca2+]i increase. Several PKCs are activated, and PKCbeta regulates phosphorylation of serine in MEKK1. Moreover, MEKKs regulate inhibitory kappaB kinase activation. Sequentially, NF-kappaB is activated, and inducible nitric-oxide synthase and tumor necrosis factor-alpha production is promoted.

Haemorrhagic shock in mice – Intracellular signalling and immunomodulation of peritoneal macrophages’ LPS response

Haemorrhagic shock leads to decreased proinflammatory cytokine response which is associated with an increased susceptibility to bacterial infections. In the present study, the effect of GM-CSF on lipopolysaccharide (LPS)-induced TNF-alpha release and MAPkinase activation was analysed on the background of a possible immunostimulating activity of this substance. Male BALB/c mice were bled to a mean arterial blood pressure of 50 mm Hg for 45 min followed by resuscitation. Peritoneal macrophages were isolated 20 h after haemorrhage and incubated with 10 ng/ml GM-CSF for 6 h before LPS stimulation. TNF-alpha synthesis was studied in the culture supernatants using ELISA. Phosphorylation of ERK, p38MAPK and IκBα was detected by Western blotting. LPS-induced TNF-alpha production of peritoneal macrophages was significantly decreased 20 h after haemorrhage in comparison to the corresponding cells of sham-operated mice. In parallel the phosphorylation of IκBα was less in LPS-stimulated peritoneal macrophages from haemorrhagic mice. LPS-induced phosphorylation of ERK1/2 was also decreased in peritoneal macrophages isolated after haemorrhagic shock. In contrast, p38MAPK was phosphorylated more intensely after LPS-stimulation in macrophages collected from shocked mice. GM-CSF incubation elevated LPS-induced TNF-alpha response of macrophages from both sham-operated and shocked mice which was accompanied by an elevated IκB and ERK phosphorylation. In general, GM-CSF treatment in vitro enhanced peritoneal macrophages LPS-response both in terms of TNF-alpha synthesis and IκB and MAPK signalling, but the levels always stayed lower than those of GM-CSF-treated cells from sham-operated animals. In conclusion, GM-CSF preincubation could partly reactivate the depressed functions of peritoneal macrophages and may therefore exert immunostimulating properties after shock or trauma.

Effect of cyclophosphamide and 61.22 GHz millimeter waves on T-cell, B-cell, and macrophage functions

The present study was undertaken to investigate whether millimeter waves (MMWs) at 61.22 GHz can modulate the effect of cyclophosphamide (CPA), an anti-cancer drug, on the immune functions of mice. During the exposure each mouse's nose was placed in front of the center of the antenna aperture (1.5 x 1.5 cm) of MMW generator. The device produced 61.22 +/- 0.2 GHz wave radiation. Spatial peak Specific Absorption Rate (SAR) at the skin surface and spatial peak incident power density were measured as 885 +/- 100 W/kg and 31 +/- 5 mW/cm(2), respectively. Duration of the exposure was 30 min each day for 3 consecutive days. The maximum temperature elevation at the tip of the nose, measured at the end of 30 min, was 1 degrees C. CPA injection (100 mg/kg) was given intraperitoneally on the second day of exposure to MMWs. The animals were sacrificed 2, 5, and 7 days after CPA administration. MMW exposure caused upregulation in tumor necrosis factor-alpha (TNF-alpha) production in peritoneal macrophages suppressed by CPA administration. MMWs also caused a significant increase in interferon-gamma (IFN-gamma) production by splenocytes and enhanced proliferative activity of T-cells. Conversely, no changes were observed in interleukin-10 (IL-10) level and B-cell proliferation. These results suggest that MMWs accelerate the recovery process selectively through a T-cell-mediated immune response.

DEHYDROEPIANDROSTERONE MODULATES TOLL-LIKE RECEPTOR EXPRESSION ON SPLENIC MACROPHAGES OF MICE AFTER SEVERE POLYMICROBIAL SEPSIS

Toll-like receptors (TLRs) play a pivotal role in the induction of innate immunity after the transactivation of proinflammatory cytokine genes. However, the responses of TLRs during severe polymicrobial sepsis have not been thoroughly examined. Although dehydroepiandrosterone (DHEA), a steroid hormone, is reported to have an immunomodulatory effect after sepsis, the mechanism responsible for its salutary is not known. To investigate this, male ICR/Jcl mice (5-8 weeks old) were subjected to sepsis by cecal ligation and puncture (CLP) or sham operation. The mice received vehicle or DHEA (40 mg/kg body weight) subcutaneously immediately after the surgery. Plasma IL-10 levels and splenic macrophage TNF-alpha production, as well as the expression levels of CD14, TLR2, and TLR4 mRNAs on splenic macrophages, were assessed 6 h after the surgery. The results indicate that mice with sepsis show a marked increase in the plasma IL-10 levels and a decrease in TNF-alpha production by splenic macrophages. TLR2 and TLR4 mRNA expression levels after CLP were significantly lower compared with those after the sham operation. TNF-alpha production and TLR2 and TLR4 mRNA expression on splenic macrophages are restored with DHEA administration. Furthermore, administration of DHEA after CLP delayed the mortality of animals. These results indicate that the anti-inflammatory phase of sepsis induces a marked down-regulation of TLR expression on splenic macrophages; however, administration of DHEA resulted in the restoration of TLR2 and TLR4 mRNA expression.

Yersinia rLcrV and rYopB inhibits the activation of murine peritoneal macrophages in vitro

Yersinia antigenic proteins LcrV and YopB are translocators of effector Yops in type III secretion system. Recently, we have reported that rLcrV and rYopB inhibit the production of TNF-alpha, IFN-gamma, and IL-12 in murine peritoneal macrophages. It was also demonstrated that IL-10 and TLR2 signaling pathways and inhibition of MAPK cascade is involved in rLcrV- and rYopB-induced immunomodulation. In the present study, it is reported that rLcrV and rYopB inhibited the LPS-induced production of IL-1beta in macrophages. Pretreatment of macrophages with rLcrV and rYopB also inhibited the LPS-induced transcription of IL-6 but not of GM-CSF. However, the transcription of chemokines like MCP-1, MIP-1alpha, MIP-1beta, and RANTES were inhibited by rLcrV and rYopB. Both proteins also affected the cytoskeleton and lipid rafts in macrophages. It is further observed that IL-10 antibodies abrogated the rLcrV- and rYopB-induced inhibition of IL-1beta production in LPS-treated macrophages. The data, therefore, suggests a possible role of IL-10 in rLcrV and rYopB mediated inhibition of LPS-induced production of IL-1beta in macrophages.

Forced retraction of spinal root injury enhances activation of p38 MAPK cascade in infiltrating macrophages

Root-rupture injury is a type of preganglionic brachial plexus injury resulting from traction force, where a small section of the spinal root is usually left behind. We have established experimental models of both root-rupture injury with traction force and rhizotomy without traction force in rats and we examined the activation of microglia/ macrophages in both conditions. LGP107 and LGP96, which are rat homologs of lysosome-associated membrane proteins, were most useful as immunohistochemical markers of mononuclear phagocytes. The metabolic activation of macrophages was analyzed by immunohistochemistry with a series of antibodies against tumor necrosis factor-alpha (TNF-alpha), cathepsin B, p38 mitogen-activated protein kinase (MAPK), and mitogen-activated kinase kinase 3 (MKK3). Both root-rupture injury and rhizotomy rapidly induced the aggregation of numerous macrophages from the injured dorsal root to the dorsal funiculus and TNF-alpha was highly expressed by the macrophages in the injured dorsal root at 48 h. Activation of p38 MAPK was preferentially observed in the macrophages at the ruptured dorsal root; however, only slight activation of p38 MAPK was observed at the rhizotomized dorsal root. These findings suggest that traction injury of the spinal root might induce activation of the p38 MAPK cascade and production of TNF-alpha in the infiltrating macrophages, both of which might participate in aggravation of the root injury.

SUPEROXIDE POTENTIATES NF-??B ACTIVATION AND MODULATES ENDOTOXIN-INDUCED CYTOKINE PRODUCTION IN ALVEOLAR MACROPHAGES

Gram-negative bacterial infection predisposes to the development of shock and acute lung injury with multiple organ dysfunction in the critically ill. Although overexpression of proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) and interleukin (IL)-1beta, IL-6, IL-8, and other mediators is causally implicated in the pathogenesis of shock and lung injury, the underlying mechanisms following cellular exposure to gram-negative endotoxin remain unclear. De novo generation of reactive oxygen species (ROS) by monocytes/macrophages in particular has been proposed as a pivotal regulatory mechanism by which enhanced transactivation of redox-sensitive genes culminates in augmented cytokine expression within the lower respiratory tract. Here we sought to characterize the mechanism of action of a synthetic, nonpeptide, low-molecular-weight, Mn-containing superoxide dismutase mimetic (SODm), M40403, in modulating E. coli lipopolysaccharide serotype 0111:B4 (LPS)-induced cytokine production by cultured rat alveolar macrophages. Intracellular superoxide (O2) ion generation was measured using hydroethidine (HE) dye, and the dose-dependent effects of M40403 on TNF-alpha and IL-6 biosynthesis by ELISAs. Upstream redox-sensitive signaling events involving the pleiotropic transcription factor NF-kappaB were determined in nuclear extracts by electrophoretic mobility shift assays (EMSAs) and p65 subunit Western blot. The levels of the cytosolic inhibitory protein IkappaB-alpha were also assessed by Western analysis. We found that M40403 potently suppressed the production of superoxide, TNF-alpha, and IL-6 in LPS-stimulated alveolar macrophages, suggesting a key role for superoxide in endotoxin-induced cytokine production in the distal air spaces. In addition, M40403 decreased E. coli LPS-induced activation of NF-kappaB, and this effect was associated with modest suppression of cytoplasmic IkappaB-alpha degradation. Together, these results suggest that removal of superoxide by M40403 inhibits endotoxin-induced production of TNF-alpha and IL-6 in alveolar macrophages by a mechanism involving suppression of redox-sensitive NF-kappaB transactivation or signaling.

Unaltered TNF-α production by macrophages and monocytes in diet-induced obesity in the rat

BackgroundRecent findings have established an association between obesity and immune dysfunction. However, most of the studies investigating the effects of obesity on immune function have been carried out in genetically obese rodent models. Since human obesity is mostly due to intake of a high fat diet and decreased energy expenditure, we asked whether immunological defects also occur in diet-induced obesity. Specifically, we focused on the function of monocytes and macrophages, as these cells are thought to be involved in the low-grade inflammation present in obesity.MethodsMale Sprague-Dawley rats were fed a high-fat or a standard chow diet for either 2 or 10 weeks. At the end of the intervention period animals were anaesthetised, blood collected for determination of plasma mediator concentrations and lipopolysaccharide (LPS) stimulated production of TNF-α by monocytes. LPS stimulated production of TNF-α in alveolar macrophages was also determined.ResultsHigh-fat feeding for either 2 or 10 weeks resulted in significant increases in fat mass and serum leptin. Although increased serum leptin has previously been linked to modulation of innate immunity, we found no significant difference in the LPS stimulated production of TNF-α by either blood monocytes or alveolar macrophages between the dietary groups. Furthermore, we failed to find a significant increase in circulating TNF-α concentrations in obese animals, as reported for genetically obese animals.ConclusionOur data suggest that defects in innate immune function observed in genetically obese animals are not mimicked by dietary obesity, and may more likely reflect the gross abnormality in leptin function of these models. Further work is required delineate the effects of dietary obesity on inflammatory state and immune function.

Inhibition of Hydrogen Peroxide, Nitric Oxide and TNF-.ALPHA. Production in Peritoneal Macrophages by Ethyl Acetate Fraction from Alchornea glandulosa

The effects of Alchornea glandulosa ethyl acetate fraction (AGF) on hydrogen peroxide (H2O2), nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha) production in peritoneal macrophages activated with lipopolysaccharide (LPS) or phorbol myristate acetate (PMA) were investigated. Analysis by thin layer chromatography (TLC) of AGF showed several constituents, including flavonoids, which may have anti-inflammatory activity. Inhibitory effects of the fraction in H2O2 and NO production ranged from 8.59+/-7.84% to 70.56+/-4.16% and from 16.06+/-3.65% to 38.73+/-3.90%, respectively. The TNF-alpha production was only partially inhibited in the tested concentrations (12.21+/-6.23% - 15.16+/-0.96%). According to these results, it is suggested that AGF has anti-inflammatory activity. This medicinal plant may have therapeutic potential in the control of inflammatory disorders.

Activation of α7 nicotinic acetylcholine receptor by nicotine selectively up-regulates cyclooxygenase-2 and prostaglandin E2 in rat microglial cultures

BackgroundNicotinic acetylcholine (Ach) receptors are ligand-gated pentameric ion channels whose main function is to transmit signals for the neurotransmitter Ach in peripheral and central nervous system. However, the α7 nicotinic receptor has been recently found in several non-neuronal cells and described as an important regulator of cellular function. Nicotine and ACh have been recently reported to inhibit tumor necrosis factor-α (TNF-α) production in human macrophages as well as in mouse microglial cultures. In the present study, we investigated whether the stimulation of α7 nicotinic receptor by the specific agonist nicotine could affect the functional state of activated microglia by promoting and/or inhibiting the release of other important pro-inflammatory and lipid mediator such as prostaglandin E2.MethodsExpression of α7 nicotinic receptor in rat microglial cell was examined by RT-PCR, immunofluorescence staining and Western blot. The functional effects of α7 receptor activation were analyzed in resting or lipopolysaccharide (LPS) stimulated microglial cells pre-treated with nicotine. Culture media were assayed for the levels of tumor necrosis factor, interleukin-1β, nitric oxide, interleukin-10 and prostaglandin E2. Total RNA was assayed by RT-PCR for the expression of COX-2 mRNA.ResultsRat microglial cells express α7 nicotinic receptor, and its activation by nicotine dose-dependently reduces the LPS-induced release of TNF-α, but has little or no effect on nitric oxide, interleukin-10 and interleukin-1β. By contrast, nicotine enhances the expression of cyclooxygenase-2 and the synthesis of one of its major products, prostaglandin E2.ConclusionsSince prostaglandin E2 modulates several macrophage and lymphocyte functions, which are instrumental for inflammatory resolution, our study further supports the existence of a brain cholinergic anti-inflammatory pathway mediated by α7 nicotinic receptor that could be potentially exploited for novel treatments of several neuropathologies in which local inflammation, sustained by activated microglia, plays a crucial role.

A serine/threonine kinase, Cot/Tpl2, modulates bacterial DNA-induced IL-12 production and Th cell differentiation.

A serine/threonine protein kinase, Cot/Tpl2, is indispensable for extracellular signal-regulated kinase (ERK) activation and production of TNF-alpha and PGE2 in LPS-stimulated macrophages. We show here that Cot/Tpl2 is also activated by other Toll-like receptor (TLR) ligands. Bacterial DNA rich in the dinucleotide CG (CpG-DNA), unlike LPS or synthetic lipopeptide, activated ERK in a Cot/Tpl2-independent manner. Peritoneal macrophages and bone marrow-derived DCs from Cot/Tpl2-/- mice produced significantly more IL-12 in response to CpG-DNA than those from WT mice. Enhanced IL-12 production in Cot/Tpl2-/- macrophages is, at least partly, regulated at the transcriptional level, and the elevated IL-12 mRNA level in Cot/Tpl2-/- macrophages is accompanied by decreased amounts of IL-12 repressors, such as c-musculoaponeurotic fibrosarcoma (c-Maf) and GATA sequence in the IL-12 promoter-binding protein (GA-12-binding protein; GAP-12) in the nucleus. Consistently, Cot/Tpl2-/- mice showed Th1-skewed antigen-specific immune responses upon OVA immunization and Leishmania major infection in vivo. These results indicate that Cot/Tpl2 is an important negative regulator of Th1-type adaptive immunity, that it achieves this regulation by inhibiting IL-12 production from accessory cells, and that it might be a potential target molecule in CpG-DNA-guided vaccination.

Arginine and glutamine availability and macrophage functions in the obese insulin-resistant Zucker rat.

Increased susceptibility to infections in obese patients may be related to decreased availability of arginine and glutamine, which may affect immune cell functions. Our aim was to evaluate the in vitro effects of these amino acids on the function of macrophages from obese insulin-resistant Zucker rats. Macrophages, isolated from male Zucker obese or lean rats by peritoneal lavage, were incubated in Dulbecco's modified Eagle medium (DMEM) without arginine or glutamine. Arginine or glutamine was added to the medium at increasing final concentrations (0, 0.25, 0.5, 1 or 2 mM). After stimulation by lipopolysaccharide (LPS) from E. coli (40 microg/ml), productions of tumour necrosis factor alpha (TNFalpha) and of nitric oxide (NO) were measured after 3 or 48 h incubation, respectively. NO production, lower in macrophages from obese rats, decreased in macrophages from lean rats (0 mM: 2,423 +/- 1,174 vs. 2 mM: 198 +/- 31 microM/mg protein/24 h; P < 0.05), but not in those from obese rats, when glutamine was added. TNFalpha production, lower in macrophages from obese rats, was inversely correlated with glutamine concentration. In the presence of arginine, NO production was constantly higher in macrophages from obese rats. It peaked at 0.5 mM arginine and decreased thereafter in both groups. TNFalpha production in macrophages from lean rats was unaffected by arginine, but decreased in macrophages from obese rats (0 mM: 1920 +/- 450 vs. 2 mM: 810 +/- 90 microM/mg protein/3 h; P < 0.05). These results suggest that abnormalities in cell signalling or in arginine and glutamine metabolism in macrophages of obese rats, resulting in decreased TNFalpha production and increased NO release, may contribute to increased susceptibility to infection in insulin-resistant states.

Adenoviral gene delivery of elafin and secretory leukocyte protease inhibitor attenuates NF-kappa B-dependent inflammatory responses of human endothelial cells and macrophages to atherogenic stimuli.

Atherosclerosis is a chronic inflammatory disease affecting arterial vessels. Strategies to reduce the inflammatory responses of endothelial cells and macrophages may slow lesion development and prevent complications such as plaque rupture. The human protease human neutrophil elastase (HNE), oxidized low density lipoprotein, LPS, and TNF-alpha were chosen as model stimuli of arterial wall inflammation and led to production of the chemokine IL-8 in endothelial cells. To counteract the activity of HNE, we have examined the effects of adenoviral gene delivery of the anti-elastases elafin, previously demonstrated within human atheroma, and murine secretory leukocyte protease inhibitor (SLPI), a related molecule, on the inflammatory responses of human endothelial cells and macrophages to atherogenic stimuli. We developed a technique of precomplexing adenovirus with cationic lipid to augment adenoviral infection efficiency in endothelial cells and to facilitate infection in macrophages. Elafin overexpression protected endothelial cells from HNE-induced IL-8 production and cytotoxicity. Elafin and murine SLPI also reduced endothelial IL-8 release in response to oxidized low density lipoprotein, LPS, and TNF-alpha and macrophage TNF-alpha production in response to LPS. This effect was associated with reduced activation of the inflammatory transcription factor NF-kappaB, through up-regulation of IkappaBalpha, in both cell types. Our work suggests a novel and extended anti-inflammatory role for these HNE inhibitors working as effectors of innate immunity to protect tissues against maladaptive inflammatory responses. Our findings indicate that elafin and SLPI may be gene therapy targets for the treatment of atheroma.

Preventive effects of a traditional Chinese medicine (Sho-saiko-to) on endotoxin-induced cytotoxicity and tumor necrosis factor-alpha production in J774A.1 Cells.

Sho-saiko-to, one of the most frequently prescribed Kampo medicines, is used clinically to treat chronic hepatitis and has shown confirmed clinical efficacy. The present study investigated whether Sho-saiko-to can suppress cytotoxicity and tumor necrosis factor (TNF)-alpha production in endotoxin-treated J774A.1 cells. Sho-saiko-to (10-20 microg/ml) did not affect the proliferation of J774A.1 cells, while a high concentration (50 microg/ml) of Sho-saiko-to induced a slight reduction in cell viability. Treatment with Sho-saiko-to (10-50 microg/ml) significantly inhibited endotoxin (10 microg/ml)-induced cytotoxicity in J774A.1 cells. In addition, Sho-saiko-to (20 microg/ml) suppressed TNF-alpha production by endotoxin (1 microg/ml)-activated J774A.1 cells. These findings suggest that the Kampo prescription Sho-saiko-to suppresses cytotoxicity or TNF-alpha production in macrophages treated with endotoxin and that it may be useful in improving septic shock symptoms. Sho-saiko-to may therefore protect against some of the various disturbances caused by endotoxins through its ability to inhibit TNF-alpha production in septic shock.

GM-CSF counteracts hemorrhage-induced suppression of cytokine-producing capacity.

To study whether a treatment with the hematopoietic growth factor GM-CSF restores the attenuated ex-vivo cytokine-producing capacity of macrophages after sublethal hemorrhagic shock. Male Sprague-Dawley rats. 20 microg/animal of recombinant murine GM-CSF after shock via arterial line. Hemorrhagic shock was established by pressure-controlled bleeding to a mean arterial pressure of 50 mm Hg for 35-40 min and consecutive resuscitation. 24 h after hemorrhage, lipopolysaccharide (LPS)-induced cytokine production of isolated macrophages derived from different compartments was measured. A significant reduction of LPS-induced TNFalpha production was found in whole blood cultures (1.0 +/- 0.7 ng/ml after sham vs. 0.23 +/- 0.08 ng/ml after shock operation), macrophages derived from spleen (0.88 +/- 0.23 ng/ml after sham vs. 0.03 +/- 0.1 ng/ml after shock operation), peritoneum (2.2 +/- 0.7 ng/ml after sham vs. 0.29 +/- 0.4 ng/ml after shock operation) and bronchoalveolar fluid (0.65 +/- 0.13 ng/ml after sham vs. 0.003 +/- 0.027 ng/ml after shock operation, mean +/- S.D.). In cells from animals treated with GM-CSF a significantly enhanced LPS-induced TNFalpha production in splenic, alveolar and peritoneal macrophages was found after shock compared to the cells derived from untreated animals (peritoneum: 289 +/- 366 ng/ml TNFalpha after shock vs. 2066 +/- 94 ng/ml TNFalpha after shock and GM-CSF; lung: 9 +/- 12 ng/ml TNFalpha after shock vs. 64 +/- 17 ng/ml TNFalpha after shock and GM-CSF; spleen: 58 +/- 96 ng/ml TNFalpha after shock vs. 548 +/- 47 ng/ml TNFalpha after shock and GM-CSF). Blood cultures collected from rats after hemorrhagic shock did not show a significant increase of TNFalpha-production after GM-CSF treatment. Hemorrhagic shock caused a depression of the TNFalphaa response to LPS which was partly counteracted by treatment with GM-CSF. Therefore, GM-CSF represents a promising approach to normalise trauma- and shock-induced immune dysfunction.

Age-dependent decrease in Toll-like receptor 4-mediated proinflammatory cytokine production and mitogen-activated protein kinase expression.

Age-related changes in immunity render elderly individuals more susceptible to infections than the young. Previous work by our laboratory and others showed that macrophages from aged mice are functionally impaired. Macrophages produce proinflammatory cytokines, tumor necrosis factor alpha (TNF-alpha) and interleukin (IL)-6, when stimulated with lipopolysaccharide (LPS), which signals through Toll-like receptor-4 (TLR4) and requires activation of mitogen-activated protein kinases (MAPKs). We investigated whether aging is associated with alterations in TNF-alpha and IL-6 production and MAPK expression and activation in thioglycollate-elicited peritoneal macrophages from mice. Kinetics and LPS dose-responsiveness of macrophage TNF-alpha production did not differ by age. Unstimulated macrophages did not differ by age in their cytokine production. However, LPS-stimulated (100 ng/mL) cultures from aged mice produced 100 +/- 30 pg/mL TNF-alpha and 6000 +/- 2000 pg/mL IL-6, and those from young mice produced 280 +/- 50 pg/mL and 10,650 +/- 10 pg/mL, respectively (P<0.05). Likewise, levels of activated MAPKs did not differ by age in unstimulated macrophages, and LPS-stimulated macrophages from aged mice had <70% activated p38 and c-jun NH(2)-terminal kinase (JNK) than those of young controls. Of particular interest, we observed >25% reduction of total p38 and JNK in macrophages from aged mice relative to young. In addition, surface TLR4 levels did not vary with age. We conclude that macrophages from aged mice exhibited suppressed proinflammatory cytokine production, which correlated with diminished total levels and LPS-stimulated activation of p38 and JNK. These observations suggest that decreased MAPK expression could be a mechanism responsible for age-related deterioration of the immune system.

Outer membrane protein 100, a versatile virulence factor of Actinobacillus actinomycetemcomitans.

Actinobacillus actinomycetemcomitans (Aa) is one of the pathogenic bacteria involved in periodontal diseases. We have previously identified six major outer membrane proteins (Omps) of Aa Y4. Among them is an Omp with high molecular mass, designated Omp100, which has homology to a variety of virulence factors. Electron microscopic observation indicated that Omp100 is randomly localized on the cell surface of Aa. Aa Y4 has been shown to adhere and invade KB or normal human gingival keratinocytes. Anti-Omp100 antibody inhibited 50% of adhesion and 70% of invasion of Aa Y4 to KB cells. An Omp100 knock-out mutant had a decreased adhesion and invasion efficiency of 60%, compared with that of the wild type. Escherichia coli HB101 expressing Omp100 adhered twofold and invaded 10-fold more than the wild-type E. coli HB101. HB101 expressing Omp100 showed resistance to serum by trapping factor H, an inhibitor for C3b, with Omp100. Omp100 induced inflammatory cytokine responses of interleukin (IL)-8, IL-6 and tumour necrosis factor (TNF)alpha in epithelial cells, and induced IL-1beta and TNFalpha production in mouse macrophages. These results indicate that Omp100 is a versatile virulence factor that may demonstrate potential significance in the onset of periodontal diseases related to Aa.

Roles of Glycosylphosphatidylinositols of Toxoplasma gondii: INDUCTION OF TUMOR NECROSIS FACTOR-α PRODUCTION IN MACROPHAGES

Toxoplasma gondii is a ubiquitous parasitic protozoan, which causes congenital infectious diseases as well as severe encephalitis, a major cause of death among immune-deficient persons, such as AIDS patients. T. gondii is normally controlled by the immune system of healthy individuals, leading to an asymptomatic infection. T. gondii triggers early cytokine production, which, to a certain extent, protects the host against replication of tachyzoites, the infective form of the parasite. Glycosylphosphatidylinositols (GPIs) constitute a class of glycolipids that have various functions, the most fundamental being to link proteins to eucaryotic cell membranes. GPIs are involved in the pathogenicity of other protozoan parasites and are known to induce tumor necrosis factor-alpha (TNF alpha) production. We show that GPIs highly purified from T. gondii tachyzoites, as well as their core glycans, induce TNF alpha production in macrophages. A chemically synthesized GPI of T. gondii lacking its lipid moiety, GPIa, has the same effect as the natural GPIs, whereas a chemically synthesized molecule with dialkylglycerol instead of diacylglycerol as lipid moiety, GPIb, does not induce TNF alpha production. Moreover, GPIb inhibits the TNF alpha production induced by T. gondii GPI or by GPIa. The core glycan prepared from the two chemically synthesized molecules activates macrophages, showing that the lipid moiety may regulate signaling. Stimulation of macrophages with GPIs of T. gondii results in activation of the transcription factor NF-kappa B, which is inhibited by the chemically synthesized GPIb, suggesting the involvement of NF-kappa B in TNF alpha gene expression. Our results support the idea that T. gondii GPIs are bioactive factors that participate in the production of TNF alpha during toxoplasmal pathogenesis.