Mannheimia (Pasteurella) haemolytica A1 produces several virulence factors that play an important role in the pathogenesis of bovine pneumonic pasteurellosis. Foremost among these is a leukotoxin (LKT) that specifically kills ruminant leukocytes. Recent evidence suggests that M. haemolytica LKT binding to bovine leukocytes is mediated by the beta(2)-integrin CD11a/CD18 (lymphocyte function-associated antigen 1 [LFA-1]), which subsequently induces activation and cytolysis of these cells. Inflammatory cytokines, which are released during viral and bacterial infection, are reported to increase LFA-1 expression and conformational activation. We investigated the effects of the inflammatory cytokines interleukin-1beta (IL-1beta), tumor necrosis factor alpha (TNF-alpha), and gamma interferon (IFN-gamma) on the interaction of M. haemolytica LKT with bovine peripheral blood neutrophils (PMNs). In this study we demonstrated, by flow cytometry, that bovine PMNs increased their binding to an anti-bovine LFA-1 monoclonal antibody (BAT75A) following in vitro incubation with IL-1beta, TNF-alpha, or IFN-gamma. Incubation with cytokines also increased CD18 expression, as assessed by real-time PCR and by Western blotting. Increased LFA-1 expression by PMNs exposed to cytokines was associated with increased LKT binding and cytotoxicity. The latter represented, at least in part, enhanced PMN apoptosis, as assessed by propidium iodine staining and caspase-3 activation. The results of this study suggest that inflammatory cytokines may play an important role in enhancing the biological response of bovine PMNs to M. haemolytica LKT.
It has been proposed that the cytokine tumor necrosis factor alpha (TNFalpha) stimulates peroxisome proliferator-induced hepatic cell proliferation. To test this hypothesis, induction of peroxisome proliferation and hepatocyte proliferation were compared in wild-type C57Bl/6 and TNFalpha knockout mice. Animals were dosed with either vehicle or 100 mg/kg/day WY14,643 by oral gavage for 4 days. Liver to brain weight ratios increased in both wild-type and TNFalpha knockout animals after WY14,643 administration. In addition, WY14,643-treated wild-type C57Bl/6 and TNFalpha knockout mice displayed marked hepatic induction of fatty acyl-CoA oxidase activity (approximately 8-fold) and mRNA content (approximately 5-fold). Electron microscopic examination confirmed increased numbers of peroxisomes in hepatocytes in both mouse models. Moreover, WY14,643 markedly induced hepatic cell proliferation (approximately 15-fold) in both wild-type C57Bl/6 and TNFalpha knockout mice as measured by bromodeoxyuridine incorporation into hepatocyte nuclei. In addition, a 50% decrease in TNFalpha mRNA was observed in wild-type mice after treatment with WY14,643. These results suggest that the hepatocellular proliferation induced after peroxisome proliferator treatment occurs independently of TNFalpha signaling.
Microbes are detected by the pathogen-associated molecular patterns through specific host pattern recognition receptors. Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) is an intracellular pattern recognition receptor that recognizes fragments of the bacterial cell wall. NOD2 is important to human biology; when it is mutated it loses the ability to respond properly to bacterial cell wall fragments. To determine the mechanisms of misactivation in the NOD2 Crohn mutants, we developed a cell-based system to screen for protein-protein interactors of NOD2. We identified heat shock protein 70 (HSP70) as a protein interactor of both wild type and Crohn mutant NOD2. HSP70 has previously been linked to inflammation, especially in the regulation of anti-inflammatory molecules. Induced HSP70 expression in cells increased the response of NOD2 to bacterial cell wall fragments. In addition, an HSP70 inhibitor, KNK437, was capable of decreasing NOD2-mediated NF-κB activation in response to bacterial cell wall stimulation. We found HSP70 to regulate the half-life of NOD2, as increasing the HSP70 level in cells increased the half-life of NOD2, and down-regulating HSP70 decreased the half-life of NOD2. The expression levels of the Crohn-associated NOD2 variants were less compared with wild type. The overexpression of HSP70 significantly increased NOD2 levels as well as the signaling capacity of the mutants. Thus, our study shows that restoring the stability of the NOD2 Crohn mutants is sufficient for rescuing the ability of these mutations to signal the presence of a bacterial cell wall ligand.
The intensity of malaria transmission is related to the pattern of malarial disease observed in different regions, but populations may also differ in their underlying predispositions to severe malarial anemia or cerebral malaria. In western Kenya, where severe malarial anemia is much more common than cerebral malaria, the distributions of tumor necrosis factor (TNF)-alpha, interleukin (IL)-10, transforming growth factor (TGF)-beta, IL-6, and interferon (IFN)-gamma alleles were examined in a cohort of young men. The cohort displayed a marked bias toward genotypes associated with low expression of IFN-gamma and IL-6, cytokines that, at high levels, have been implicated in malarial anemia and poor malaria outcomes. By contrast, the frequency of the TNF-alpha -238A allele, which has been associated with severe malarial anemia, was found to be similar to the frequency previously reported in comparison populations in Africa and elsewhere. IFN-gamma and IL-6 genotypes may play roles in the development of severe malaria and could contribute to the relative frequency of severe malarial anemia or cerebral malaria in exposed populations.
The correction of anemia by recombinant human erythropoietin (rHuEPO) improves quality of life and prolongs life in end-stage renal failure. rHuEPO requirements for an individual are determined by a range of factors, including iron deficiency and inflammation. Single nucleotide polymorphisms in the promoter sequence of several proinflammatory cytokines have been shown, in different fields of medicine, to influence the cytokine response to different stimuli, with effects on clinical outcome.
The angiotensin-converting enzyme (ACE) insertion/deletion polymorphism and polymorphisms in the promoter regions of the genes for tumor necrosis factor alpha (-308 A/G), interleukin-6 (-174 G/C), and interferon gamma were examined for their association with rHuEPO requirements in 112 patients on continuous ambulatory peritoneal dialysis (CAPD). Genomic DNA was extracted from peripheral blood leukocytes and genotyping performed with ARMS-PCR methodology, with sequence-specific primers. We examined rHuEPO requirements and C-reactive protein at baseline and during a 6-month study period.
We found no significant effect of proinflammatory cytokine polymorphisms on rHuEPO responsiveness. However, throughout the study, we observed that there was a significantly higher rHuEPO requirement in the II and ID ACE genotypes compared with the DD group, which remained an independent association following multivariate analysis.
ACE insertion/deletion polymorphism may determine rHuEPO responsiveness in CAPD patients and should be considered in relative rHuEPO resistance.
TNF and its receptors p55 and p75 are known to be important in the homeostasis of the peripheral immune system. Previous studies have presented apparently contradictory evidence for an in vivo role of TNF in T cells. In this study, we analyzed TNF-deficient mice crossed with the F5 TCR-transgenic animals. We show that endogenous TNF modulates several aspects of homeostasis of peripheral F5 CD8 T cells. We found that F5/TNF(-/-)mice had reduced numbers of peripheral F5 T cells, F5/TNF(-/-) CD8 T cells exhibited reduced survival potential, and furthermore that T cell-derived TNF is required for optimum recovery of naive CD8 T cells in lymphopenic hosts, suggesting its involvement in the survival of peripheral CD8 T cells. Both peptide activation and ensuing Ag-induced apoptosis are quantitatively reduced in TNF(-/-) CD8 T cells. The latter observations can be related to decreased binding activities of NF-kappaB and NF-ATp observed in Ag-stimulated F5/TNF(-/-) T cells. Finally, in a CD8 T cell tolerance model, endogenous TNF was necessary for several parameters of CD8 T cell tolerance induction. Collectively, our results provide evidence that endogenous TNF modulates thresholds in several ligand-driven T cell responses.
The roles of free fatty acids (FFA), tumor necrosis factor-alpha (TNF-alpha), and adiponectin in the development of the insulin-resistant metabolic disorder in several subjects have been studied. A total of 70 Japanese male subjects were selected according to the following three sets of criteria: subjects in group A had, (1) a fasting plasma glucose (FPG)>or=110 to <140 mg/dl, (2) a triglyceride (TG) level>or=150 mg/dl, (3) a systolic blood pressure (SBP)>or=140 and/or diastolic blood pressure (DBP)>or=90 mmHg, and (4) a body mass index (BMI)>or=25 kg/m2 (age=53.4+/-8.5 years, BMI=27.0+/-1.3 kg/m2, n=16). Subjects in group B had, (1) FPG<110 mg/dl, (2) TG<150 mg/dl, (3) SBP<140 and DBP<90 mmHg, and (4) BMI>or=25 kg/m2 (age=47.2+/-10.3 years, BMI=26.6+/-1.31 kg/m2, n=38). Subjects in group C had, (1) FPG<110 mg/dl, (2) TG<150 mg/dl, (3) SBP<140 and DBP<90 mmHg, and (4) 20>or=BMI<22 kg/m2 (age=50.4+/-9.3 years, BMI=20.9+/-0.6 kg/m2, n=16). The homeostasis model assessment of insulin resistance in group A (2.7+/-1.4) was significantly higher (p<0.0001) than in groups B (1.6+/-0.7) and C (0.9+/-0.5). FFA in group A (1.17+/-0.57 mEq/l) was significantly higher than in groups B (0.62+/-0.23 mEq/l) and C (0.48+/-0.16 mEq/l) (p<0.0001). Serum TNF-alpha in group A (1.36+/-0.62 pg/ml) was significantly higher than in groups B (0.95+/-0.35 pg/ml; p=0.003) and C (0.76+/-0.09 pg/ml; p=0.0013). No significant differences in the serum level of adiponectin were observed between groups A and B or between groups B and C. The results suggest that FFA and possibly TNF-alpha levels are closely related to the development of insulin resistance in subjects with metabolic disorders.
Cell line studies have previously demonstrated that hypoxia-reoxygenation (H/R) leads to the production of NADPH oxidase 1 and 2 (NOX1 and NOX2)-dependent reactive oxygen species (ROS) required for the activation of c-Src and NF-κB. We now extend these studies into mouse models to evaluate the contribution of hepatocytes to the NOX- and c-Src-dependent TNF-α production that follows H/R in primary hepatocytes and liver ischemia-reperfusion (I/R). In vitro, c-Src-deficient primary hepatocytes produced less ROS and TNF-α following H/R compared with controls. In vivo, c-Src-KO mice also had impaired TNF-α and NF-κB responses following partial lobar liver I/R. Studies in NOX1 and p47phox knockout primary hepatocytes demonstrated that both NOX1 and p47phox are partially required for H/R-mediated TNF-α production. To further investigate the involvement of NADPH oxidases in the production of TNF-α following liver I/R, we performed additional in vivo experiments in knockout mice deficient for NOX1, NOX2, p47phox, Rac1, and/or Rac2. Cumulatively, these results demonstrate that NOX2 and its activator subunits (p47phox and Rac) control the secretion of TNF-α by the liver following I/R. Interestingly, in the absence of Kupffer cells and NOX2, NOX1 played a dominant role in TNF-α production following hepatic I/R. However, NOX1 deletion alone had little effect on I/R-induced TNF-α. Thus Kupffer cell-derived factors and NOX2 act to suppress hepatic NOX1-dependent TNF-α production. We conclude that c-Src and NADPH oxidase components are necessary for redox-mediated production of TNF-α following liver I/R and that hepatocytes play an important role in this process.
Cytokine-mediated inflammation is important in the pathogenesis of non-small cell lung cancer (NSCLC). Genetic polymorphisms in cytokine genes and their association with lung cancer in the Indian population have not been reported.
For the first time, we analyzed genetic polymorphisms of TNFα -308, IFNγ +874, and IL10 -1082 genes in 246 NSCLC patients and 250 healthy controls in the South Indian population from Telangana using ARMS PCR.
IFNγ+874 A/T and IL10-1082 G/G gene polymorphisms were found to be significantly associated with NSCLC with 1.56- and 1.68-fold disease risk, respectively. There was no association between the risk of NSCLC and TNFα-308 polymorphism. Gene polymorphisms stratified according to smoking revealed that IFNγ+874 A/T polymorphisms in smokers increased the disease risk by 2.91 fold. IL10-1082 G/G polymorphisms showed 2-fold increased risk among patients who were smokers when compared to the controls. However, there was no association between TNFα-308, IFNγ+874, and IL10-1082 gene polymorphism and the stage of the NSCLC patients. The overall risk associated with the combination of these polymorphisms indicated that the TNFα-308 G/A + IFNγ+874 A/T + IL10-1082 G/G genotype increased the risk by 1.5 fold.
The results of our study indicate an association between cytokine gene polymorphisms and the risk of NSCLC in an Indian population.
This study evaluates the effects of the gold nanoparticle in endotoxin-induced uveitis in rats.
Adult male Wistar rats were divided into five groups: saline + saline, lipopolysaccharide (LPS) + saline, LPS + prednisolone, LPS + gold salt (GS) and LPS + gold nanoparticle (GNP). Two hours after LPS administration, prednisolone acetate 1%, GS, and GNP were topically applied to both eyes of rats and repeated every 6 hours for 24 hours. After 24 hours, rats were anesthetized and aqueous humor was sampled and the irides were removed. Aqueous humor TNF-α, myeloperoxidase activity were determined. Irides oxidative damage and content of toll-like receptor 4 (TLR4) and nuclear factor-κB (NF-κB) were determined.
The administration of LPS-induced eye inflammatory response characterized by an increase in aqueous humor TNF-α, myeloperoxidase, and by irides oxidative damage. All these parameters were decreased by the administration of GNP. Since the inflammatory response secondary to LPS administration depends, in part, to the activation of the TLR4-NF-κB pathway we demonstrated here that a potential mechanism to explain the GNP effects was the decrease on TLR4 content and NF-κB activation.
These findings suggest that topical GNP decreases intraocular inflammation and oxidative damage by interfering in the TLR4-NF-κB pathway.