The gut cytokine balance as a target of lead toxicity.

The impact of exposure to lead on gut cytokine gene expression and oral tolerance was analyzed. Oral tolerization with ovalbumin (OVA) increased levels of IL-10 and TGF-beta in gut tissue while IFN-gamma mRNA levels remained unchanged in both autoimmune diabetes prone NOD and normal C57BL/6 mice. This shift towards Th2/Th3 type cytokine gene expression was completely abolished by concomitant treatment with PbCl2 (6 x 0.5 mg/kg) in NOD mice while the cytokine balance in C57BL/6 mice was unaffected. Suppression of Th2/Th3 type cytokine expression was associated with a dampened oral tolerance response to OVA as determined by T cell proliferation assays. We conclude that in autoimmunity prone NOD mice environmental toxicants may disturb immune homeostasis by targeting the gut immune system.     

Inhibitory effect of dietary n-3 polyunsaturated fatty acids to intestinal IL-15 expression is associated with reduction of TCRalphabeta+CD8alpha+CD8beta-intestinal intraepithelial lymphocytes

Intestinal intraepithelial lymphocytes (IELs) and their cytokines play an important role in the regulation of gut immune response and take part in gut immune barrier function. n-3 polyunsaturated fatty acid (PUFA) is an immunoregulator that has been shown to influence the process of gut inflammation. Interleukin (IL)-15 is a T-cell growth factor that has been shown to influence the differentiation of IEL. The aim of this study was to analyze the effects of dietary n-3 PUFA on IEL. IEL phenotype and cytokine (TNF-alpha, IFN-gamma, IL-4, IL-10 and TGF-beta1) profile were measured by FACS and real-time RT-PCR in healthy adult rats fed with fish oil diet for 90 days. Rats fed with corn oil diet served as controls. Intestinal IL-15 expression was measured by immunohistochemistry and real-time RT-PCR. The results demonstrated a decrease of intestinal IL-15 expression in the fish oil group. Associated with this deduction, n-3 PUFA significantly decreased the proportion of TCRalphabeta+CD8alpha+CD8beta- cells and IEL-derived TNF-alpha, IFN-gamma, IL-4 and IL-10. In conclusion, n-3 PUFA could inhibit intestinal mucosal expression of IL-15 and may influence phenotype and function of IEL through this mechanism.     

Immunoregulation during disease progression in prediabetic NOD mice: inverse expression of arginase and prostaglandin H synthase 2 vs. interleukin-15.

Non-obese diabetic (NOD) mice spontaneously develop insulin dependent diabetes due to autoimmune destruction of beta-cells. The progression of insulitis can be accelerated and synchronized in the pancreas by a single injection of 250 mg/kg cyclophosphamide. In this study, we will report on three immune mediators that were not known to be expressed during insulitis until now. Early insulitis in ten-week-old female NOD mice was associated with strong expression of prostaglandin H synthase 2 in the pancreas and of arginase, an antagonist enzyme of the inducible NO synthase. After acceleration of insulitis progression by cyclophosphamide, expression of the two enzymes was downregulated within 24 h. There was strong concomitant upregulation of IL-15 gene expression that preceded lymphocyte invasion of islets and a rise of IFN-gamma mRNA levels by several days. The comparison of individual pancreata showed that the expression of IL-12 and IL-18 mRNA closely correlated with levels of IL-15 gene expression. We conclude that arginase and prostaglandin H synthase 2 expression is associated with peri-insulitis, while IL-15 is a candidate cytokine in driving destructive insulitis, as it elicits Th1-cytotoxic responses in lymphoid as well as in non-lymphoid immune cells and is unusually resistant to downregulation by antagonistic cytokines. This is the first report on arginase, prostaglandin H synthase 2 and IL-15 expression in pancreatic lesions of prediabetic NOD mice.     

Nitric oxide synthesis and TNF-alpha secretion in RAW 264.7 macrophages: mode of action of a fermented papaya preparation

Macrophage inducible nitric oxide synthase is able to generate massive amounts of nitric oxide (NO) which contributes to the host immune defense against viruses and bacteria. Monocyte-macrophages stimulated with the bacterial wall component lipopolysaccharide (LPS) and cytokines such as interferon-gamma (IFN-gamma) express the inducible form of nitric oxide synthase (iNOS). Furthermore, tumor necrosis factor-alpha (TNF-alpha) is one of the central regulatory cytokines in macrophage antimicrobial activity and synergizes with IFN-gamma in the induction of NO synthesis. Because of its pivotal role in both antimicrobial and tumoricidal activities of macrophages, a significant effort has focused on developing therapeutic agents that regulate NO production. In the present study fermented papaya preparation (FPP) is shown to exert both immunomodulatory and antioxidant activity in the macrophage cell line RAW 264.7. Interestingly, a low and a high molecular weight fraction (LMF and HMF, respectively) of FPP exhibited different activity patterns. FPP fractions alone did not affect NO production. However in the presence of IFN-gamma, both LMF and HMF significantly increased iNOS activity and nitrite as well as nitrate accumulation. NO radical formation measured in real-time by electron paramagnetic resonance spectroscopy was higher in the presence of LMF and IFN-gamma. On the contrary, iNOS mRNA levels were enhanced further with HMF than with LMF. Moreover, LMF displayed a stronger superoxide anion scavenging activity than HMF. In the presence of IFN-gamma, both FPP fractions stimulated TNF-alpha secretion. However in non-stimulated macrophages, TNF-alpha secretion was enhanced by HMF only. Since water-soluble FPP fractions contained no lipid A, present data indicate that FPP is a macrophage activator which augments nitric oxide synthesis and TNF-alpha secretion independently of lipopolysaccharides.     

Prevention of Cyclophosphamide-induced Accelerated Diabetes in the NOD Mouse by Nicotinamide or a Soy Protein-based Infant Formula

Spontaneous diabetes in the NOD mouse can be prevented by nicotinamide or by an infant formula diet in which the protein source is replaced with casein hydrolysate (Pregestimil) or soy protein (Prosobee). NOD mice maintained on the standard diet (chow and water) and given cyclophosphamide (Cy) at day 95 develop accelerated and synchronised diabetes within 14 days. Here, we compared the ability of oral nicotinamide or Prosobee, either given alone or concurrently, from weaning, in preventing diabetes in the Cy model. The resulting insulitis and the expression of intra-islet inducible nitric oxide synthase (iNOS) were examined at days 0, 4, 7, 11 and 14 following Cy administration. Intra-islet CD4 and CD8 cells and macrophages were also enumerated at day 11. In mice given the standard diet and injected with Cy at day 95 (group 5), diabetes developed in 7/11 mice, 14 days later. Mice exposed to oral nicotinamide (group 2), Prosobee (group 3) or both (group 4), did not develop the disease during this period and until a further 30 days (p = 0.03). In mice exposed to the standard diet and without Cy treatment (group 1) the insulitis scores increased slowly until day 11 and then declined slightly at day 14 whereas mice exposed to the same diet but given Cy at day 95, showed a sharp decline at day 4 followed by a rapid increase between day 7–14. However, in mice given either nicotinamide, Prosobee or both, the insulitis scores at most time-points were generally lower than in Cy-teated animals on the standard diet. In the latter group, CD4 and CD8 cells and macrophages were also higher at day 11 than all other 4 groups (CD4: p < 0.05; CD8: p < 0.05; macrophages: p < 0.0001). The number of iNOS labelled cells increased progressively in mice on the standard diet and given Cy and were significantly higher at days 4, 7 and 11 than in the 3 dietary groups. Thus, oral nicotinamide or Prosobee, either alone or together, prevents Cy induced diabetes in the NOD mouse. The protective diets suppress Cyinduced intra-islet immune cell influx and iNOS expression.     

TGF-beta1 enhances degradation of IFN-gamma-induced iNOS protein via proteasomes in RAW 264.7 cells.

TGF-beta1 is a well-known immunosuppressive cytokine that inhibits inducible nitric oxide synthase (iNOS) gene expression in various cells including macrophages. In this study, we investigated the suppressive mechanisms of TGF-beta1 on IFN-gamma-induced iNOS gene expression using the murine macrophage-like cell line RAW 264.7. TGF-beta1 decreased iNOS protein amount through enhanced degradation, although TGF-beta1 did not affect IFN-gamma-induced iNOS mRNA level or stability. In addition, the enhancement of iNOS protein degradation by TGF-beta1 treatment was almost completely blocked by MG132, a proteasome inhibitor. Furthermore, TGF-beta1 enhanced the trypsin-like activity of proteasomes in the presence of IFN-gamma, although did not enhance the peptidylglutamyl-peptide hydrolyzing and chymotrypsin-like activities of proteasomes. The level of ubiquitinated iNOS protein was not significantly altered by IFN-gamma or IFN-gamma plus TGF-beta1 treatment. Because MG132 inhibited iNOS protein degradation and IFN-gamma plus TGF-beta1 treatment increased the trypsin-like activity of proteasomes, we hypothesized that TGF-beta1 might enhance iNOS protein degradation via the ubiquitin-proteasome pathway in the presence of IFN-gamma. We propose that these mechanisms of TGF-beta1 in the posttranslational regulation of iNOS gene expression may contribute to suppression of excess nitric oxide during inflammatory processes.     

Molecular mechanisms of high-dose antigen therapy in experimental autoimmune encephalomyelitis: rapid induction of Th1-type cytokines and inducible nitric oxide synthase

High-dose Ag administration induces apoptotic death of autoreactive T cells and is an effective therapy of experimental autoimmune diseases of the nervous system. To explore the role of cytokines in Ag-specific immunotherapy, we analyzed mRNA induction and protein expression for the proinflammatory cytokines TNF-alpha and IFN-gamma, the anti-inflammatory cytokine IL-10, and the cytokine-inducible NO synthase (iNOS) during high-dose Ag therapy of adoptive transfer experimental autoimmune encephalomyelitis (AT-EAE) in the Lewis rat. Using semiquantitative and competitive RT-PCR, we found 5- to 6-fold induction of TNF-alpha mRNA and 3-fold induction of IFN-gamma mRNA in the spinal cord that occurred within 1 h after i.v. injection of Ag and was accompanied by a 2-fold increase of iNOS mRNA. Both IFN-gamma and iNOS mRNA remained elevated for at least 6 h, whereas TNF-alpha mRNA was already down-regulated 6 h after Ag injection. A comparable time course was found for circulating serum levels of TNF-alpha and IFN-gamma. IL-10 mRNA levels did not change significantly following Ag injection. Neutralization of TNF-alpha by anti-TNF-alpha antiserum in vivo led to a significant decrease in the rate of T cell and oligodendrocyte apoptosis induced by high-dose Ag administration, but did not change the beneficial clinical effect of Ag therapy. Our data suggest profound activation of proinflammatory but not of anti-inflammatory cytokine gene expression by high-dose Ag injection. Functionally, TNF-alpha contributes to increased apoptosis of both autoaggressive T cells and oligodendrocytes in the target organ and may thereby play a dual role in this model of Ag-specific therapy of CNS autoimmune diseases.     

Inhibition of inducible NO synthase by TH2 cytokines and TGF beta in rheumatoid arthritic synoviocytes: effects on nitrosothiol production.

The aim of this study was to compare the effects on NO production of IL-4, IL-10, and IL-13 with those of TGF-beta. RA synovial cells were stimulated for 24 h with IL-1 beta (1 ng/ml), TNF-alpha (500 pg/ml), IFN-gamma (10(-4)IU/ml) alone or in combination. Nitrite was determined by the Griess reaction, S-nitrosothiols by fluorescence, and inducible NO synthase (iNOS) by immunofluorescence and fluorescence activated cell sorter analysis (FACS). In other experiments, IL-4, IL-10, IL-13, and TGF beta were used at various concentrations and were added in combination with proinflammatory cytokines. The addition of IL-1 beta, TNF-alpha, and IFN-gamma together increased nitrite production: 257.5 +/- 35.8 % and S-nitrosothiol production : 413 +/- 29%, P < 0.001. None of these cytokines added alone had any significant effect. iNOS synthesis increased with NO production. IL-4, IL-10, IL-13, and TGF beta strongly decreased the NO production caused by the combination of IL-1 beta, TNF-alpha, and IFN-gamma. These results demonstrate that stimulated RA synoviocytes produce S-nitrosothiols, bioactive NO* compounds, in similar quantities to nitrite. IL-4, IL-10, IL-13, and TGF-beta decrease NO production by RA synovial cells. The anti-inflammatory properties of these cytokines may thus be due at least in part to their effect on NO metabolism.     

Regulation of the RON receptor tyrosine kinase expression in macrophages: blocking the RON gene transcription by endotoxin-induced nitric oxide

Previous studies have shown that activation of the RON receptor tyrosine kinase inhibits inducible NO production in murine peritoneal macrophages. The purpose of this study is to determine whether inflammatory mediators such as LPS, IFN-gamma, and TNF-alpha regulate RON expression. Western blot analysis showed that RON expression is reduced in peritoneal macrophages collected from mice injected with a low dose of LPS. The inhibition was seen as early as 8 h after LPS challenge. Experiments in vitro also demonstrated that the levels of the RON mRNA and protein are diminished in cultured peritoneal macrophages following LPS stimulation. TNF-alpha plus IFN-gamma abrogated macrophage RON expression, although individual cytokines had no significant effect. Because LPS and TNF-alpha plus IFN-gamma induce NO production, we reasoned that NO might be involved in the RON inhibition. Two NO donors, S-nitroglutathione (GSNO) and (+/-)-S-nitroso-N-acetylpenicillamine (SNAP), directly inhibited macrophage RON expression when added to the cell cultures. Blocking NO production by NO inhibitors like TGF-beta prevented the LPS-mediated inhibitory effect. In Raw264.7 cells transiently transfected with a report vector, GSNO or SNAP inhibited the luciferase activities driven by the RON gene promoter. Moreover, GSNO or SNAP inhibited the macrophage-stimulating protein-induced RON phosphorylation and macrophage migration. We concluded from these data that RON expression in macrophages is regulated during inflammation. LPS and TNF-alpha plus IFN-gamma are capable of down-regulating RON expression through induction of NO production. The inhibitory effect of NO is mediated by suppression of the RON gene promoter activities.     

Inducible nitric oxide synthase mediates cytokine release: the time course in conscious and septic rats

Nitric oxide (NO), tumor necrosis factor-alpha (TNF-alpha), and interleukin 1-beta (IL-1beta) are postulated to play a key pathophysiologic role during sepsis. In this study, we examined the time course of inducible NO synthase (iNOS) mRNA expression and the plasma TNF-alpha and IL-1beta in lipopolysaccharide (LPS)-treated conscious rats. The hemodynamic pattern in septic shock is more similar to clinical conditions without anesthesia. The data showed that a significant increase in iNOS mRNA levels was found in the spleen, lung, liver, with slight elevation in the heart and kidney at 3 h after LPS administration. However, iNOS mRNA levels were not elevated significantly in all tissues examined at 24 h. In the plasma, TNF-alpha and IL-1beta culminated within 1 h, and reduced gradually to baseline levels in a relatively short period (within 9 h). The results suggest that local NO production by activation of iNOS mRNA expression and cytokine release may contribute to LPS-induced organ dysfunction at various time points.     

Mechanisms of spontaneous resolution versus fibrosis in granulomatous experimental autoimmune thyroiditis

When granulomatous experimental autoimmune thyroiditis (G-EAT) was induced in CBA/J or DBA/1 mice, thyroid lesions resolved in less severe (3+) G-EAT in wild-type mice or severe (5+) G-EAT in IFN-gamma(-/-) mice, but progressed to fibrosis in 5+ G-EAT in wild-type mice. To define the mechanisms leading to these distinct outcomes, the expression of inflammatory and apoptotic molecules and infiltrating cells was evaluated using immunohistochemistry, RT-PCR, and confocal microscopy. The ratio of CD4(+)/CD8(+) T cells in thyroid infiltrates was one factor that predicted G-EAT outcome. CD4(+) T cells outnumbered CD8(+) T cells when lesions progressed to fibrosis, while CD8(+) T cells outnumbered CD4(+) T cells in thyroids that resolved. Fas, Fas ligand, FLIP, TNF-alpha, inducible NO synthase, TGF-beta, and IFN-gamma were highly expressed by infiltrating cells when G-EAT progressed to fibrosis. The expression of active caspase-3 was low, possibly contributing to the persistence of CD4(+) T cells in fibrosis. In contrast, FLIP was mainly expressed by thyrocytes in resolving G-EAT, the expression of active caspase-3 was high, and resolution correlated with apoptosis of infiltrating cells. There was also relatively less expression of TGF-beta, IFN-gamma, TNF-alpha, and inducible NO synthase and higher expression of IL-10 in resolving G-EAT than in G-EAT that progressed to fibrosis. These differences were particularly striking when comparing IFN-gamma(-/-) vs wild-type mice. These results suggest that several opposing biological mechanisms contribute to the outcome of an ongoing autoimmune response. These include differential expression of pro- and antiapoptotic molecules, cytokines, and the ratio of CD4(+) vs CD8(+) T cells.     

Macrolide antibiotics protect against immune complex-induced lung injury in rats: role of nitric oxide from alveolar macrophages.

Macrolide antibiotics have unique immunomodulatory actions apart from antimicrobial properties. We studied the effects of macrolides on IgG immune complex (IgG-ICx)-induced lung injury in rats in vivo and in vitro. Intrapulmonary deposition of IgG-ICx produced a time-dependent increase in the concentration of NO in exhaled air. There were corresponding increases in the number of neutrophils accumulated into alveolar spaces, and lung wet-to-dry weight ratio. All of these changes were inhibited by pretreatment with erythromycin or josamycin, but not by amoxicillin or cephaclor. Incubation of cultured pulmonary alveolar macrophages caused up-regulation of NO production and expression of inducible NO synthase mRNA, an effect that was dose dependently inhibited by erythromycin, roxithromycin, or josamycin. The macrolides also reduced IgG-ICx-induced release of IL-1beta and TNF-alpha, but did not alter the release of NO induced by exogenously added IL-1beta and TNF-alpha. These results suggest that macrolide antibiotics specifically inhibit immune complex-induced lung injury presumably by inhibiting cytokine release and the resultant down-regulation of inducible NO synthase gene expression and NO production by rat pulmonary alveolar macrophages.     

Mechanisms underlying resistance of pancreatic islets from ALR/Lt mice to cytokine-induced destruction

Nuclear and mitochondrial genomes combine in ALR/Lt mice to produce systemically elevated defenses against free radical damage, rendering these mice resistant to immune-mediated pancreatic islet destruction. We analyzed the mechanism whereby isolated islets from ALR mice resisted proinflammatory stress mediated by combined cytokines (IL-1beta, TNF-alpha, and IFN-gamma) in vitro. Such damage entails both superoxide and NO radical generation, as well as peroxynitrite, resulting from their combination. In contrast to islets from other mouse strains, ALR islets expressed constitutively higher glutathione reductase, glutathione peroxidase, and higher ratios of reduced to oxidized glutathione. Following incubation with combined cytokines, islets from control strains produced significantly higher levels of hydrogen peroxide and NO than islets from ALR mice. Nitrotyrosine was generated in NOD and C3H/HeJ islets but not by ALR islets. Western blot analysis showed that combined cytokines up-regulated the NF-kappaB inducible NO synthase in NOD-Rag and C3H/HeJ islets but not in ALR islets. This inability of cytokine-treated ALR islets to up-regulate inducible NO synthase and produce NO correlated both with reduced kinetics of IkappaB degradation and with markedly suppressed NF-kappaB p65 nuclear translocation. Hence, ALR/Lt islets resist cytokine-induced diabetogenic stress through enhanced dissipation and/or suppressed formation of reactive oxygen and nitrogen species, impaired IkappaB degradation, and blunted NF-kappaB activation. Nitrotyrosylation of beta cell proteins may generate neoantigens; therefore, resistance of ALR islets to nitrotyrosine formation may, in part, explain why ALR mice are resistant to type 1 diabetes when reconstituted with a NOD immune system.