Increased TNF-alpha Secretion Research Articles

Quercetin suppresses inflammation by reducing ERK1/2 phosphorylation and NF kappa B activation in Leptin-induced Human Umbilical Vein Endothelial Cells (HUVECs)

BackgroundHigh concentrations of plasma leptin and the release of pro-inflammatory cytokines in leptin-resistance in obesity have been reported to trigger endothelial dysfunction. The objective of this study was to elucidate the role of quercetin in modulating leptin-induced inflammation as assessed by the levels of Ob-Ra expression, ERK1/2 phosphorylation, NF-kappa B activation and TNF-alpha secretion in umbilical vein endothelial cells (HUVECs) in vitro.FindingsHUVECs were exposed to either control levels (0 ng/ml) or 500 ng/mL leptin (L) for 48 hours, followed by control or 125 uM quercetin (Q) for another 6 h. The experimental groups were as follows: L0Q0, L0Q125, L500Q0, L500Q125. The presence of the short chain leptin receptor isoform Ob-Ra in HUVECs was determined by Western blot and immunocytochemistry analyses. Ob-Ra expression, ERK1/2 phosphorylation, NF-kappa B activation and TNF-alpha secretion were quantified by ELISA, and NF-kappa B activationby immunofluorescence staining. Our results showed that Ob-Ra expression, ERK1/2 phosphorylation and NF-kappa B activation increased significantly after 500 ng/mL leptin exposure (1.8x, 1.5x, 6.2x for Ob-Ra, ERK1/2 and NF-kappa B, respectively), but were reduced by addition of 125 uM quercetin (0.7x, 0.3x and 0.4x for Ob-Ra, ERK1/2 and NF-kappa B, respectively), and that quercetin could also partially suppress leptin-induced TNF-alpha secretion (3.8x) by 0.8x.ConclusionExposure of HUVECs to leptin up-regulated Ob-Ra expression and elevated ERK1/2 phosphorylation and NFkB activation, and increased TNF-alpha secretion. These effects strongly suppressed by quercetin, with the exception of TNF-alpha which was partially suppressed. The findings might be of clinical significance, as endothelial dysfunction that could lead to cardiovascular disease is preventable, and quercetin is a natural compound found in various plants and fruits.

Alcohol depletes coenzyme-Q10 associated with increased TNF-alpha secretion to induce cytotoxicity in HepG2 cells

Alcohol consumption has been implicated to cause severe hepatic steatosis which is mediated by alcohol dehydrogenase (ADH) activity and CYP450 2E1 expression. In this context, the effect of ethanol was studied for its influence on lipogenesis in HepG2 cell which is deficient of ADH and does not express CYP450 2E1. The results showed that ethanol at 100mM concentration caused 40% cytotoxicity at 72h as determined by MTT assay. The incorporation of labeled [2-14C] acetate into triacylglycerol and phospholipid was increased by 40% and 26% respectively upon 24h incubation, whereas incorporation of labeled [2-14C] acetate into cholesterol was not significantly increased. Further, ethanol inhibited HMG-CoA reductase which is a rate-limiting enzyme in the cholesterol biosynthesis. It was observed that, HMG-CoA reductase inhibition was brought about by ethanol as a consequence of decreased cell viability, since incubation of HepG2 cells with mevalonate could not increase the cholesterol content and increase the cell viability. Addition of ethanol significantly increased TNF-alpha secretion and depleted mitochondrial coenzyme-Q10 which is detrimental for cell viability. But vitamin E (10mM) could partially restore coenzyme-Q10 and glutathione content with decreased TNF-alpha secretion in ethanol treated cells. Further, lipid peroxidation, glutathione peroxidase and superoxide dismutase enzyme activities remained unaffected. Ethanol decreased glutathione content while, GSH/GSSG ratio was significantly higher compared to other groups showing cellular pro-oxidant and antioxidant balance remained intact. Alanine amino transferase activity was increased by 4.85 folds in cells treated with ethanol confirming hepatocyte damage. Hence, it is inferred that ethanol induced cytotoxicity in HepG2 cells due to coenzyme-Q10 depletion and increased TNF-alpha secretion.

Extended LTA, TNF, LST1 and HLA Gene Haplotypes and Their Association with Rubella Vaccine-Induced Immunity

BackgroundRecent studies have suggested the importance of HLA genes in determining immune responses following rubella vaccine. The telomeric class III region of the HLA complex harbors several genes, including lymphotoxin alpha (LTA), tumor necrosis factor (TNF) and leukocyte specific transcript -1 (LST1) genes, located between the class I B and class II DRB1 loci. Apart from HLA, little is known about the effect of this extended genetic region on HLA haplotypic backgrounds as applied to immune responses.Methodology/Principal FindingsWe examined the association between immune responses and extended class I-class II-class III haplotypes among 714 healthy children after two doses of rubella vaccination. These extended haplotypes were then compared to the HLA-only haplotypes. The most significant association was observed between haplotypes extending across the HLA class I region, ten-SNP haplotypes, and the HLA class II region (i.e. A-C-B-LTA-TNF-LST1-DRB1-DQA1-DQB1-DPA1-DPB1) and rubella-specific antibodies (global p-value of 0.03). Associations were found between both extended A*02-C*03-B*15-AAAACGGGGC-DRB1*04-DQA1*03-DQB1*03-DPA1*01-DPB1*04 (p = 0.002) and HLA-only A*02-C*03-B*15-DRB1*04-DQA1*03-DQB1*03-DPA1*01-DPB1*04 haplotypes (p = 0.009) and higher levels of rubella antibodies. The class II HLA-only haplotype DRB1*13-DQA1*01-DQB1*06-DPA1*01-DPB1*04 (p = 0.04) lacking LTA-TNF-LST1 SNPs was associated with lower rubella antibody responses. Similarly, the class I-class II HLA-only A*01-C*07-B*08-DRB1*03-DQA1*05-DQB1*02-DPA1*01-DPB1*04 haplotype was associated with increased TNF-α secretion levels (p = 0.009). In contrast, the extended AAAACGGGGC-DRB1*01-DQA1*01-DQB1*05-DPA1*01-DPB1*04 (p = 0.01) haplotype was found to trend with decreased rubella-specific IL-6 secretion levels.Conclusions/SignificanceThese data suggest the importance of examining both HLA genes and genes in the class III region as part of the extended haplotypes useful in understanding genomic drivers regulating immune responses to rubella vaccine.

Bacterial components regulate the expression of Toll-like receptor 4 on human mast cells

The aim of this study was to investigate whether the exposure of mast cells (MCs) to bacterial components affects the expression of Toll-like receptor (TLR) 4, and to elucidate the behavior of MCs during the early response to infection. Two human MC lines, HMC-1 and LAD2, were employed. Messenger RNA expression was observed by RT and real-time PCR. TLR4 expression was determined by Western blotting. TNF-alpha secretion was analyzed with ELISA. The degranulation ratio was measured with betahexosaminidase assay. Although bacterial components increased TLR4 mRNA, only lipopolysaccharide (LPS) augmented the TLR4 protein expression. LAD2 pre-treated with LPS for 8 h resulted in 2-fold increased TNF-alpha secretion on LPS restimulation. These results suggest that the exposure of MCs to LPS may reinforce the innate immune system due to up-regulation of MC TLR4, followed by increased TNF-alpha release.

Kruppel-like factor 5 is an important mediator for lipopolysaccharide-induced proinflammatory response in intestinal epithelial cells

Lipopolysaccharide (LPS) is a bacterially-derived endotoxin that elicits a strong proinflammatory response in intestinal epithelial cells. It is well established that LPS activates this response through NF-κB. In addition, LPS signals through the mitogen-activated protein kinase (MAPK) pathway. We previously demonstrated that the Krüppel-like factor 5 [KLF5; also known as intestine-enriched Krüppel-like factor (IKLF)] is activated by the MAPK. In the current study, we examined whether KLF5 mediates the signaling cascade elicited by LPS. Treatment of the intestinal epithelial cell line, IEC6, with LPS resulted in a dose- and time-dependent increase in KLF5 messenger RNA (mRNA) and protein levels. Concurrently, mRNA levels of the p50 and p65 subunits of NF-κB were increased by LPS treatment. Pretreatment with the MAPK inhibitor, U0126, or the LPS antagonist, polymyxin B, resulted in an attenuation of KLF5, p50 and p65 NF-κB subunit mRNA levels from LPS treatment. Importantly, suppression of KLF5 by small interfering RNA (siRNA) resulted in a reduction in p50 and p65 subunit mRNA levels and NF-κB DNA binding activity in response to LPS. LPS treatment also led to an increase in secretion of TNF-α and IL-6 from IEC6, both of which were reduced by siRNA inhibition of KLF5. In addition, intercellular adhesion molecule-1 (ICAM-1) levels were increased in LPS-treated IEC6 cells and this increase was associated with increased adhesion of Jurkat lymphocytes to IEC6. The induction of ICAM-1 expression and T cell adhesion to IEC6 by LPS were both abrogated by siRNA inhibition of KLF5. These results indicate that KLF5 is an important mediator for the proinflammatory response elicited by LPS in intestinal epithelial cells.

JNK and Tumor Necrosis Factor-α Mediate Free Fatty Acid-induced Insulin Resistance in 3T3-L1 Adipocytes

Lipid infusion and high fat feeding are established causes of systemic and adipose tissue insulin resistance. In this study, we treated 3T3-L1 adipocytes with a mixture of free fatty acids (FFAs) to investigate the molecular mechanisms underlying fat-induced insulin resistance. FFA treatment impaired insulin receptor-mediated signal transduction and decreased insulin-stimulated GLUT4 translocation and glucose transport. FFAs activated the stress/inflammatory kinases c-Jun N-terminal kinase (JNK) and IKKbeta, and the suppressor of cytokine signaling protein 3, increased secretion of the inflammatory cytokine tumor necrosis factor (TNF)-alpha, and decreased secretion of adiponectin into the medium. RNA interference-mediated down-regulation of JNK blocked JNK activation and prevented most of the FFA-induced defects in insulin action. Blockade of TNF-alpha signaling with neutralizing antibodies to TNF-alpha or its receptors or with a dominant negative TNF-alpha peptide had a partial effect to inhibit FFA-induced cellular insulin resistance. We found that JNK activation by FFAs was not inhibited by blocking TNF-alpha signaling, whereas the FFA-induced increase in TNF-alpha secretion was inhibited by RNA interference-mediated JNK knockdown. Together, these results indicate that 1) JNK can be activated by FFAs through TNF-alpha-independent mechanisms, 2) activated JNK is a major contributor to FFA-induced cellular insulin resistance, and 3) TNF-alpha is an autocrine/paracrine downstream effector of activated JNK that can also mediate insulin resistance.

Tumor necrosis factor alpha secretion by peritoneal macrophages in patients with endometriosis.

As a cytotoxic product of activated monocytes, macrophages, and lymphocytes, tumor necrosis factor alpha (TNF-alpha)--together with other cytokines and growth factors--is an important component in the immune response of the human organism. In addition, TNF-alpha plays a central role in neoangiogenesis. Because of its cytotoxicity with regard to several tumor cells and its motility-hindering effect on human sperm, TNF-alpha is considered to be a significant pelvic mediator of female sterility. The goal of our study was to determine as to whether or not an increased TNF-alpha secretion by peritoneal macrophages (PM) can be measured in female patients with endometriosis compared with healthy subjects, and if TNF-alpha secretion can be correlated with the activity of endometriosis. During infertility work-up, 100 female patients underwent a diagnostic laparoscopy. In accordance with the rAFS classification as well as from the macroscopic aspect of the degree of activity of the endometriosis, the patients were divided as follows: an endometriosis-free control group with a completely normal pelvic status (n=35) and three groups with increasing stages of endometriosis (n=65). In the control group (Group 1), the TNF-alpha concentrations (median values with minimum / maximum) were 6.2 pg/ml (1.9/10.2), in Group 2 with rAFS stage I/II less active endometriosis 56.33 pg/ml (39.5/71.2), in Group 3 with rAFS stage I/II but highly active endometriosis 81.41 pg/ml (68.4/98.7), while in Group 4 with rAFS stage III/IV 200,15 pg/ml (182.6/226.8), respectively. In conclusion, we were able to show that the TNF-alpha secretion of PM was significantly higher in patients with proven endometriosis compared to the control group. These results were found to be statistically significant and were in accordance with the histological findings. Thus, due to its immunomodulating potential, TNF-alpha may be a marker of both activity and stage of endometriosis.

Identification of meningococcal LPS as a major monocyte activator in IL-10 depleted shock plasmas and CSF by blocking the CD14-TLR4 receptor complex.

We have examined the in vitro stimulatory effects of lipopolysaccharide (LPS)-containing samples (meningococcal shock plasma, n = 10; non-shock plasma, n = 10; cerebrospinal fluid (CSF), n = 7) before and after immunodepletion of interleukin (IL)-10 in a monocyte target assay. We also studied the stimulatory effects of plasma collected from 3 patients with lethal septicemia caused by Streptococcus pneumoniae without detectable LPS but with 100-fold increased levels of heat-shock protein 70 (HSP70). HSP70 may, like LPS, activate monocytes via the Toll-like receptor 4 (TLR4). The samples were analyzed for LPS, tumor necrosis factor (TNF)-alpha, IL-10 and HSP70; applied on human monocytes (purity > 95%) before and after IL-10 immunodepletion, in the absence or presence of CD14 blocking mAb (60bca) or the lipid A antagonist, Rhodobacter sphaeroides diphosphoryl lipid A (RsDPLA) which blocks TLR4. Monocyte activation was measured by increased TNF-alpha secretion and tissue factor (TF) up-regulation by monocyte procoagulant activity (PCA). There was a positive correlation between patient plasma LPS levels (n = 10) and increases in TNF-alpha secretion by the monocytes after immunodepletion of IL-10 (r = 0.82). Pretreatment of the monocytes with mAbCD14 or RsDPLA reduced TNF-alpha secretion to median 5% and 12%, respectively, of the levels before the receptor complex was blocked. The median levels of HSP70 were 543 ng/ml (range, 468-962 ng/ml) in pneumococcal shock plasma, 81.5 ng/ml (range, 41-331 ng/ml) in meningococcal shock plasma and 24 ng/ml (range, < 0.8-41 ng/ml) in meningococcal non-shock plasma. Pneumococcal septic shock plasmas with significantly higher levels of HSP70 (P < 0.05) did not induce TNF-alpha secretion in the monocytes. The results strongly suggest that LPS in meningococcal shock plasma is the major activator of monocytes whereas HSP70 (in plasma concentrations up to 963 ng/ml) does not activate monocytes in this assay.

Characterization of the immune response against hepatitis C infection in recovered, and chronically infected chimpanzees.

The immune response to hepatitis C virus (HCV) is believed to be critical in determining the outcome of the disease. In this study we have analysed epitope recognition, cytokine profile, and anti-HCV antibody responses in chronically HCV-infected and recovered chimpanzees. Quantitative measurement of anti-HCV antibody in HCV-infected chimpanzees revealed that the response in HCV- recovered chimpanzees peaked within 4-20 weeks. In contrast, the anti-HCV antibody responses in chronically HCV infected chimpanzees did not peak until 100-200 weeks after infection, and decreased gradually thereafter. T cell proliferation assays measuring responses to pooled HCV proteins revealed significant increases in the 3H-uptake during the early stages of infection in recovered chimpanzees in comparison to the chronically infected ones. Class I-restricted epitopes of the core, and NS3 proteins of HCV were analysed using 9-10 mer overlapping peptides covering the core and NS3 proteins, and IFN-gamma ELISPOT technique. Our data indicated early and broad class-I restricted core, and NS3 protein epitope recognitions in HCV-recovered chimpanzees but not in chimpanzees that had been chronically infected. Additionally, dominant epitopes recognized early in infection (8 weeks) were no longer recognized later in infection (followed up to 64 weeks). Cytokines profiling revealed a 50-fold increase in TNF-alpha secretion in the supernatant of core-specific CD8 memory cells of the chronically infected chimpanzees in comparison to the recovered ones. In summary, multiple parameters correlate with HCV recovery in chimpanzees.

Activation of stress-responsive pathways by the sympathetic nervous system in burn trauma.

We have shown previously that bum trauma activates the stress responsive proteins, p38 mitogen-activated protein kinase (MAPK), c-jun NH2-terminal kinase (JNK), and NF-kappaB, and we have shown further that p38 MAPK is an important mediator of cardiomyocyte TNF-alpha secretion and cardiac dysfunction in burn trauma. Since burn trauma causes a rise in circulating catecholamine levels, we hypothesized that this increased sympathetic activity may function as an upstream activator of the p38 MARK pathway in burn trauma. This study determined whether the alpha1-adrenergic receptor ligand phenylephrine could mimic burn trauma activation of p38 MAPK, JNK, and NF-kappaB nuclear translocation; and the effect of the alpha1-adrenergic receptor antagonist prazosin on either phenylephrine or burn-mediated activation of the stress response pathway was examined. Sprague Dawley rats were divided into seven groups: Group 1, controls; Group 2, phenylephrine-treated (2 microg/kg, i.v.) control rats; Group 3, phenylephrine-treated plus prazosin-treated (1 mg/kg, i.v.) control rats; additional rats were given burn over 40% total body surface area (TBSA) and received vehicle (1 mL of 2% sucrose, p.o.) plus fluid resuscitation (Group 4), while in Group 5, burn rats were given prazosin (1 mg/kg, p.o.) plus fluid resuscitation. In Groups 6 and 7, sham-burned rats were given either vehicle (1 mL of 2% sucrose, p.o.) or prazosin (1 mg/kg, p.o.) to provide appropriate controls. Administration of phenylephrine to rats caused a significant activation of cardiac p38 MAPK/JNK activities (Western blot) and cardiac NF-kappaB nuclear translocation (electrophoretic mobility shift assay, EMSA). Prazosin blocked phenylephrine mediated changes in p38 MAPK/JNK activities. Burn trauma activated cardiac p38 MAPK/JNK and NF-kappaB, increased TNF-alpha secretion by cardiomyocytes, and impaired cardiac function. Prazosin treatment in burns interrupted the burn-mediated signaling cascade, decreasing TNF-alpha secretion by cardiomyocytes and preventing post-burn cardiac contractile dysfunction. Thus, burn trauma-related sympathetic activity likely activates the stress-responsive cascade, which regulates myocardial TNF-alpha transcription/translation and culminates in cardiac contraction and relaxation defects.

The Time Course of Cardiac NF-??B Activation and TNF-?? Secretion by Cardiac Myocytes After Burn Injury: Contribution to Burn-Related Cardiac Contractile Dysfunction

Previous studies have suggested that cardiac synthesis of TNF-alpha contributes to myocardial dysfunction in several models of trauma, sepsis and ischemia. Therefore, it is likely that myocyte secretion of TNF-alpha occurs early after major burn trauma, contributing to progressive cardiac contractile dysfunction that is characteristic of thermal injury. This study examined the time course of nuclear translocation of the transcription factor NF-kappaB, the time course of TNF-alpha secretion by cardiomyocytes after burn trauma, and the development of cardiac contractile defects. Rats were given burn injury over 40% TBSA (sham burns included for controls), and fluid resuscitation included lactated Ringer's solution, 4 mL/kg/%burn. Subsets of rats were sacrificed at several times postburn (1, 2, 4, 8, 12, 18 and 24 h), hearts were harvested to prepare cardiomyocytes (N = 4 rats/group/time period), to prepare nuclear fractions to measure burn-induced NF-kappaB activation (N = 3-4 rats/group/time period), or to examine the time course of postburn cardiac contractile dysfunction (N = 6-7 rats/group/time period). Despite aggressive fluid resuscitation, burn trauma activated NF-kappaB 4 h postburn, and this activation persisted over the 24 h study period. In addition, burn trauma produced a time-related increase in TNF-alpha secretion by cardiac myocytes with cytokine secretion evident 1 h postburn. Cardiac dysfunction occurred 8 h postburn and persisted over the 24 h study period. Administration of a strategy designed to inhibit NF-kappaB activation (N-acetyl-leucinyl-leucinyl-norleucinal, ALLN, 50 mg/kg, in additional groups of burn rats) inhibited TNF-alpha secretion by cardiac myocytes and improved myocardial function. This study confirms that burn trauma activates myocardial NF-kappaB and promotes cardiomyocyte secretion of TNF-alpha. This inflammatory cascade preceded the appearance of cardiac dysfunction, suggesting that cardiac myocyte derived TNF-alpha contributes, in part, to postburn cardiac contractile deficits.

Lipopolysaccharide induces expression of genes encoding pro-inflammatory cytokines and the elastin-degrading enzyme, cathepsin S, in human cervical smooth-muscle cells.

Vaginal and amniotic infection with gram-negative bacteria is associated with preterm birth. We previously reported that human cervical smooth-muscle cells (CSMC) respond to pro-inflammatory cytokines by expressing enzymes that degrade the extracellular matrix. Our objective was to characterize the effects of lipopolysaccharide (LPS) from Escherichia coli (E coli), Bacteroides fragilis, (B frag)and Fusobacterium nucleatum (F nuc)on the expression of pro-inflammatory cytokines and the elastin-degrading enzyme, cathepsin S, in human CSMC. Human CSMC were exposed to LPS and the expression of mRNAs encoding pro-inflammatory cytokines and cathepsin S, and selected matrix metalloproteinases (MMPs) was analyzed by Northern blotting. The effect of cytokine-neutralizing antibodies on LPS-induced cathepsin S mRNA expression also was determined. E coli LPS increased expression of cathepsin S 12.5-fold after 12 hours; MMP-1 and MMP-3 mRNAs also were increased 2.9- and 3.5-fold, respectively. Tumor necrosis factor (TNF)-alpha, interleukin (IL-1)alpha, and IL-1beta mRNAs were markedly up-regulated after 3 hours of LPS treatment. B frag and F nuc LPS also induced TNF-alpha and cathepsin S mRNAs. E coli LPS caused a sevenfold increase in TNF-alpha secretion after 5 to 8 hours. Antihuman TNF-alpha monoclonal antibody, but not a monoclonal antibody to the low-density lipoprotein receptor, reduced the LPS-induced increase in cathepsin S mRNA by 27%, whereas neutralizing antibodies against IL-1alpha and IL-1beta did not suppress the response. Human CSMC were shown to express the toll-like receptor (TLR-2) and TLR-4 genes, which mediate the action of LPS. TLR-2 mRNA was up-regulated by TNF-alpha. CSMC respond to LPS with increased expression of pro-inflammatory cytokines and cathepsin S. Increases in cathepsin S mRNA result only in part from the rapid induction of TNF-alpha gene expression. TNF-alpha may also augment the CSMC response to LPS by increasing expression of the LPS signaling receptor, TLR-2, which probably directly mediates LPS action. These observations provide a mechanism by which gram-negative bacteria can precipitate cervical changes associated with preterm birth.

Mdr1 promoter-driven tumor necrosis factor-alpha expression for a chemotherapy-controllable combined in vivo gene therapy and chemotherapy of tumors.

Cancer gene therapy approaches are often designed as single-agent treatments; however, greater therapeutic effect might be obtained if combined with an established conventional treatment regimen such as chemotherapy. In this context, conditional promoters are useful tools, because they may be induced by therapeutic modalities. The human multidrug resistance gene (mdr1) promoter is inducible by cytostatic drugs and can be employed for the chemotherapy-regulated expression of therapeutic genes. In this in vivo study, the human mdr1 promoter fragment (-207 to +158) was used for drug-inducible expression of human tumor necrosis factor-alpha (TNF-alpha) in the vector construct pM3mdr-p-hTNF. The single doxorubicin and vincristine treatment of nude mice xenografted with pM3mdr-p-hTNF-transduced MCF-7 mammary tumors resulted in drug-induced and time-dependent elevation of intratumoral TNF-alpha expression at the mRNA and protein level. The highest drug induction was achieved at 2 days after drug application, as reflected by a maximum 25-fold increase in TNF-alpha secretion in the tumor. This drug-induced TNF-alpha expression is more effective in inhibiting tumor growth compared with the growth of tumors transduced with constitutively TNF-alpha-expressing vectors in combination with chemotherapy.

Increase of proliferation rate and enhancement of antitumor cytotoxicity of expanded human CD3+ CD56+ immunologic effector cells by receptor-mediated transfection with the interleukin-7 gene.

Cytokine-induced killer (CIK) cells have been shown to eradicate established tumors in a SCID mouse-human lymphoma model. CIK cells depend on exogenous addition of cytokines such as interleukin-2 (IL-2), interleukin-7 (IL-7) or interleukin-12 (IL-12) for proliferation. In this study, we used the adenovirus-enhanced CD3 receptor-mediated gene transfer for transfection with the IL-7 gene. An episomally replicating plasmid was used containing cDNA of the human IL-7 gene under the control of a CMV promoter for transfection of CIK cells. Biosynthesis of IL-7 was demonstrated by RT-PCR, an enzyme-linked immunosorbent assay (ELISA) and using a bioassay. Transfected cells produced IL-7 in the range between 200 and 1100 pg/10(6) cells in 24 h. IL-7 was shown to be biologically active, since transfected CIK cells showed an improved proliferation rate as compared with nontransfected cells. Expression of IL-7 altered the secretion of other cytokines by CIK cells, in particular the production of TNF alpha increased after transfection. In contrast, nontransfected CIK cells fed with IL-7 showed no increase in TNF alpha secretion. No significant differences were found in expression of surface antigens linked to the cytotoxic activity of CIK cells. Cytotoxic activity against various tumor cell lines (eg renal cell carcinoma, malignant melanoma and colon carcinoma) was tested. Transfected cells possessed a significantly higher cytotoxic activity as compared with nontransfected cells. Receptor-mediated gene transfer effectively delivers expression plasmids for therapeutic genes into CIK cells and CIK cells transfected with an IL-7 gene expression construct may be valuable for adoptive immunotherapy.

Leukocyte-polytetrafluoroethylene interaction enhances proliferation of vascular smooth muscle cells via tumor necrosis factor-α secretion

Intimal hyperplasia of vascular smooth muscle cells (VSMC) at the venous anastomosis of arteriovenous grafts represents the most common cause of vascular access failure in hemodialysis patients. Upstream release of growth factors from leukocytes activated by adhesion to the graft material may play a role in this lesion. We evaluated the effect of interaction of peripheral blood mononuclear cells (PBMC) with polytetrafluoroethylene (PTFE) on proliferation of VSMC. Vascular smooth muscle cell proliferation was significantly increased by conditioned media from human PBMC incubated with PTFE. Peripheral blood mononuclear cell adhesion to PTFE could not be antagonized by the beta 1 integrin ligand-containing peptide GRGDSP, but was attenuated by EDTA consistent with beta 2 integrin-mediated adhesion. Soluble scavenger receptor ligands at high concentrations had no effect on adhesion to PTFE excluding any contributory role of scavenger receptors in this interaction. Neutralizing antibodies to TNF-alpha significantly attenuated the mitogenic effect of PBMC/PTFE conditioned media and a marked increase in TNF-alpha secretion by PBMC on PTFE was detected by ELISA. These studies demonstrate that PBMC interaction with PTFE can promote proliferation of VSMC via increased production of TNF-alpha and perhaps other cytokines. Leukocyte interaction with PTFE causing enhanced secretion of TNF-alpha and consequent VSMC proliferation may account for the development of venous intimal hyperplasia in hemodialysis patients with arteriovenous grafts.

The heavy metal lead modulates the expression of both TNF-alpha and TNF-alpha receptors in lipopolysaccharide-activated human peripheral blood mononuclear cells.

It has been shown that lead (Pb) potentiates lipopolysaccharide (LPS) lethality in animals by increasing the secretion and uptake or reactivity of tumor necrosis factor-alpha (TNF-alpha). Herein we report that PbCl2 increased TNF-alpha secretion from LPS-treated human peripheral blood mononuclear cells (PBMC) in a concentration- and time-dependent manner. PbCl2 also increased total cellular TNF-alpha levels but had no effect on the steady-state levels of TNF-alpha mRNA. PbCl2 decreased membrane-associated TNF-alpha (mTNF-alpha) on LPS-treated monocytes, whereas PbCl2 increased TNF-alpha receptor (TNF-R) p55 surface expression, and had no effect on TNF-R p75 surface expression by LPS-treated monocytes. Overall, the results suggest that PbCl2 increases TNF-alpha expression by posttranscriptional mechanisms in human PBMC, and enhances the reactivity and uptake of TNF-alpha by increasing the surface expression of TNF-R p55.

Role of defective monocyte interleukin-10 release in tumor necrosis factor-alpha overproduction in alcoholic cirrhosis

Monocytes of patients with alcoholic cirrhosis produce higher amounts of tumor necrosis factor-alpha (TNF-alpha) after lipopolysaccharide (LPS) stimulation. The mechanisms of the overproduction remain undefined. IL-10 (IL-10) is an antiinflammatory cytokine known to downregulate TNF-alpha secretion by monocytes. The present study analyzes IL-10 production by monocytes and its control on TNF-alpha secretion in alcoholic cirrhosis. LPS-stimulated monocytes from alcoholic cirrhotics (n = 13) showed decreased IL-10 (median, 240 pg/mL [40 to 500] upsilon 513 pg/mL [152 to 1,335]; P = .01) compared with controls (n = 13). Cells from cirrhotic patients were normally responsive to recombinant IL-10, which induced a dose dependent decrease of TNF-alpha secretion. On the other hand, preincubation with anti-IL-10 monoclonal antibodies led to significant increase in TNF-alpha secretion in controls (median, 7,325 to 16,800 pg/mL; P = .002) but not in cells from cirrhotic patients (16,535 to 20,450 pg/mL; P = .14), abolishing the difference in TNF-alpha production between cirrhotic patients and controls. It is concluded that defective IL-10 secretion by monocytes from alcoholic cirrhotic patients could be involved in the characteristics TNF-alpha overproduction observed in this disease.

Ozone stimulates synthesis of inflammatory cytokines by alveolar macrophages in vitro.

Ozone (O3) is one of the major irritant oxidant gases in photochemical smog. In the present study, the in vitro effect of low concentrations of O3 (0.1 to 1 ppm) was evaluated on cell viability and cytokine secretion by alveolar macrophages (AM) from guinea pigs and healthy subjects. Cell injury was estimated immediately after O3 exposure by evaluation of ATP cell content (measured by bioluminescence) and lactic dehydrogenase (LDH) release in the culture medium. No cytotoxic effect was found: the ATP cell content of both guinea pig AM and human AM did not significantly change after O3 exposure and similarly the LDH release in the culture medium was unchanged. AM-derived cytokines (tumor necrosis factor alpha [TNF alpha], interleukin-1 beta [IL-1 beta], interleukin-6 [IL-6], and interleukin-8 [IL-8]) were evaluated in AM supernatants. O3 exposure was associated with a significant increase in cytokine secretion, with a peak value at 0.4 ppm O3. The exposure of the guinea pig AM to 0.4 ppm O3 for 60 min increased the IL-6 activity by 252 +/- 60% and TNF activity by 202 +/- 35%. The increase in monokine production by the human AM was 443 +/- 208% for TNF alpha, 484 +/- 171% for IL-1 beta, 383 +/- 147% for IL-6, and 226 +/- 45% for IL-8 after a 60-min exposure to 0.4 ppm O3. Lowest O3 concentrations (0.1 and 0.2 ppm) only increased TNF alpha secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Porphyromonas gingivalis lipopolysaccharide stimulation of human monocytes: dependence on serum and CD14 receptor

The purpose of this study was to investigate factors influencing the ability of lipopolysaccharide (LPS) derived from Porphyromonas gingivalis to elicit secretion of tumor necrosis factor-alpha (TNF alpha) from human monocytes (adherent mononuclear cells). The results indicate that P. gingivalis LPS stimulation of TNF alpha from monocytes is comparable to LPS from Escherichia coli. Both LPS, although structurally different, increased TNF alpha secretion in a dose-dependent manner. In serum-free conditions, TNF alpha secretion was relatively low, but it dramatically increased at human serum concentrations as low as 1%. Maximal secretion was observed in the presence of 10% serum, with a slight decrease at higher serum concentrations. The CD14 molecule is a putative monocyte LPS receptor. When cells were pre-incubated with a blocking monoclonal antibody (My4) to CD14, TNF alpha-mRNA accumulation and TNF alpha secretion were reduced to control levels at LPS concentrations of up to 10 ng/ml. At higher LPS concentrations, the blocking effect was only partial, in spite of 50-fold excess antibody concentration. The blocking effect was observed only in the presence of serum. The effect of the CD14 antibody was dose-dependent with saturation at 2.5 micrograms/ml. The results suggest that CD14 is one of the major receptors for P. gingivalis LPS but highlight the necessity to investigate other cell-surface receptors mediating P. gingivalis-LPS interactions. These interactions are believed to be important in the pathogenesis of periodontal destruction.

Substance P enhances the secretion of tumor necrosis factor-alpha from neuroglial cells stimulated with lipopolysaccharide.

The neuropeptide, substance P (SP), can stimulate secretion of TNF-alpha from macrophages. Neuroglia have SP receptors and subserve various macrophage-like functions in the central nervous system. We investigated whether SP stimulates secretion of TNF-alpha from primary cultures of neuroglial cells containing both astrocytes (approximately 90%) and microglia (approximately 10%). SP alone had no effect; however in the presence of LPS (10 ng/ml), SP (1 to 10 nM) caused a dose-dependent increase in TNF-alpha secretion above the level measured in response to LPS alone. The effective doses of SP correlated with 125I-labeled Bolton Hunter-conjugated SP binding (Kd 0.2 nM) to these cultures. Incubation with LPS did not change the number or affinity of SP-binding sites. In cultures enriched for microglia (> 99% pure), LPS stimulated the secretion of TNF-alpha but SP caused no enhancement. Microglia have no detectable 125I-labeled Bolton-Hunter-conjugated SP binding sites in the presence or absence of LPS. These results indicate that the action of SP is mediated through astrocytes. We investigated whether IL-1 mediates the SP enhancement of TNF-alpha secretion. Addition of IL-1-neutralizing antisera to mixed cultures stimulated with both LPS and 10 nM SP decreased TNF-alpha secretion to the level observed with LPS alone. LPS alone stimulated the secretion of IL-1 in a dose-dependent manner in the primary cultures, and this LPS-mediated IL-1 secretion was enhanced by SP. This enhancement was not observed in microglial cultures. SP may therefore play a role in neuropathologies in which these cytokines have been implicated.