Micronucleus studies in the peripheral blood and bone marrow of mice treated with jet fuels, JP-8 and Jet-A

The potential adverse effects of dermal and inhalation exposure of jet fuels are important for health hazard evaluation in humans. The genotoxic potential of jet fuels, JP-8 and Jet-A, was investigated in an animal model. Mice were treated dermally with either a single or multiple applications of these jet fuels. Peripheral blood and bone marrow smears were prepared to examine the incidence of micronuclei (MN) in polychromatic erythrocytes (PCEs). In all experiments, using several different exposure regimens, no statistically significant increase in the incidence of MN was observed in the bone marrow and/or peripheral blood of mice treated with JP-8 or Jet-A when compared with those of untreated control animals. The data in mice treated with a single dose of JP-8 or Jet-A did not confirm the small but statistically significant increase in micronuclei reported in our previous study.     

Effect of proinflammatory cytokines on interleukin-8 mRNA expression and protein production by isolated human alveolar epithelial cells type II in primary culture

Alveolar epithelial cells type II (AEC-II) are ideally situated to regulate the recruitment and activation of different types of cells through the production of chemokines in response to inflammatory stimulation from the alveolar space. We hypothesized that these cells are important producers of interleukin-8 (IL-8) in the lung. This lead us to investigate the capacity of isolated human AEC-II cells to release IL-8 and whether this IL-8 release is regulated by proinflammatory cytokines, i.e. IL-1 beta, TNF-alpha and IFN-gamma. We isolated AEC-II from tumor-free sections of human lungs obtained by pneumectomy and purified the cells by magnetic activated cell sorting. For control experiments the AEC-II-like cell line A549 was used. IL-8 concentration was measured by ELISA in supernatants of unstimulated and LPS-, IL-1 beta-, TNF-alpha- and IFN-gamma- stimulated cells. IL-8 mRNA expression was evaluated by RT-PCR. Spontaneous IL-8 mRNA expression and protein secretion by AEC-II were significantly higher in comparison with A549 cells. TNF-alpha increased both IL-8 mRNA expression and protein production, whereas IL-1 beta slightly increased IL-8 release but did not change mRNA expression in AEC-II. LPS and IFN-gamma did not influence IL-8 expression in AEC-II and A549 cells. These results show considerable differences between A549 cell and AEC-II. The latter are capable of producing IL-8 under the control of proinflammatory cytokines. Our findings demonstrate that the modulation of IL-8 release in AEC-II may have an important impact on the immunoreactivity of these cells during pulmonary inflammation in vivo.     

Cytogenetic studies in mice treated with the jet fuels, Jet-A and JP-8

The genotoxic potential of the jet fuels, Jet-A and JP-8, were examined in mice treated on the skin with a single dose of 240 mg/mouse. Peripheral blood smears were prepared at the start of the experiment (t = 0), and at 24, 48 and 72 h following treatment with jet fuels. Femoral bone marrow smears were made when all animals were sacrificed at 72 h. In both tissues, the extent of genotoxicity was determined from the incidence of micronuclei (MN) in polychromatic erythrocytes. The frequency of MN in the peripheral blood of mice treated with Jet-A and JP-8 increased over time and reached statistical significance at 72 h, as compared with concurrent control animals. The incidence of MN was also higher in bone marrow cells of mice exposed to Jet-A and JP-8 as compared with controls. Thus, at the dose tested, a small but significant genotoxic effect of jet fuels was observed in the blood and bone marrow cells of mice treated on the skin.     

Candida albicans and Streptococcus salivarius modulate IL-6, IL-8, and TNF-alpha expression and secretion by engineered human oral mucosa cells

We investigated the involvement of oral epithelial cells via two cytokines (IL-6 and TNF-alpha) and one chemokine (IL-8) in local defences against live yeast (Candida albicans) and bacteria (Streptococcus salivarius) using an engineered human oral mucosa model. We report that the yeast changed from the blastospore to the hyphal form and induced significant tissue disorganization at later contact periods (24 and 48 h) compared to the bacteria. However, this effect did not reduce the viability or total number of epithelial cells. Gene activation analyses revealed that IL-6, IL-8 and TNF-alpha mRNA levels rose in tissues in contact with live C. albicans or S. salivarius. Gene activation was followed by an upregulation of protein secretion. IL-6 levels were higher after contact with C. albicans than with S. salivarius. IL-8 levels after contact with S. salivarius were higher than with C. albicans. Our study suggests that S. salivarius is more efficient at inducing proinflammatory mediator release than C. albicans. These results provide additional evidence for the contribution of oral epithelial cells to the inflammatory response against fungi and bacteria.     

LPS and proinflammatory cytokines decrease lipin-1 in mouse adipose tissue and 3T3-L1 adipocytes

Infection and inflammation affect adipose triglyceride metabolism, resulting in increased plasma free fatty acid (FFA) and VLDL levels during the acute-phase response. Lipin-1, a multifunctional protein, plays a critical role in adipose differentiation, mitochondrial oxidation, and triglyceride synthesis. Here, we examined whether LPS [a Toll-like receptor (TLR)-4 activator], zymosan (a TLR-2 activator), and proinflammatory cytokines regulate lipin-1 in adipose tissue. LPS administration caused a marked decrease in the levels of lipin-1 mRNA and protein in adipose tissue. The decrease in lipin-1 mRNA levels occurred rapidly and lasted for at least 24 h. In contrast, lipin-2 and -3 mRNA levels did not change, suggesting specific repression of lipin-1. Zymosan similarly decreased lipin-1 mRNA without affecting lipin-2 or lipin-3 mRNA levels. To determine the pathways by which LPS repressed lipin-1, we examined the effect of proinflammatory cytokines on cultured adipocytes. In 3T3-L1 adipocytes, TNF-alpha, IL-1beta, and IFN-gamma, but not LPS or IL-6, caused a decrease in lipin-1 mRNA levels. Furthermore, TNF-alpha and IL-1beta administration also decreased mRNA levels of lipin-1 in adipose tissue in mice. Importantly, the LPS-induced decrease in lipin-1 mRNA levels was significantly but not totally blunted in TNF-alpha/IL-1 receptor-null mice compared with controls, suggesting key roles for TNF-alpha/IL-1beta and other cytokines in mediating LPS-induced repression of lipin-1. Together, our results demonstrate that expression of lipin-1, one of the essential triglyceride synthetic enzymes, was suppressed by LPS, zymosan, and proinflammatory cytokines in mouse adipose tissue and in cultured 3T3-L1 adipocytes, which could contribute to a decrease in the utilization of FFA to synthesize triglycerides in adipose tissue, thus promoting the release of FFA into the circulation.     

The new IL-1 family member IL-1F8 stimulates production of inflammatory mediators by synovial fibroblasts and articular chondrocytes

Six novel members of the IL-1 family of cytokines were recently identified, primarily through the use of DNA database searches for IL-1 homologues, and were named IL-1F5 to IL-1F10. In the present study, we investigated the effect of IL-1F8 on primary human joint cells, and examined the expression of the new IL-1 family members in human and mouse joints. Human synovial fibroblasts (hSFs) and human articular chondrocytes (hACs) expressed the IL-1F8 receptor (IL-1Rrp2) and produced pro-inflammatory mediators in response to recombinant IL-1F8. IL-1F8 mRNA expression was increased in hSFs upon stimulation with proinflammatory cytokines, whereas in hACs IL-1F8 mRNA expression was constitutive. However, IL-1F8 protein was undetectable in hSF and hAC culture supernatants. Furthermore, although IL-1beta protein levels were increased in inflamed human and mouse joint tissue, IL-1F8 protein levels were not. IL-1F8 levels in synovial fluids were similar to or lower than those in matched serum samples, suggesting that the joint itself is not a major source of IL-1F8. Serum levels of IL-1F8 were similar in healthy donors, and patients with rheumatoid arthritis, osteoarthritis and septic shock, and did not correlate with inflammatory status. Interestingly however, we observed high IL-1F8 levels in several serum samples in all groups. In conclusion, IL-1F8 exerts proinflammatory effects in primary human joint cells. Joint and serum IL-1F8 protein levels did not correlate with inflammation, but they were high in some human serum samples tested, including samples from patients with rheumatoid arthritis. It remains to be determined whether circulating IL-1F8 can contribute to joint inflammation in rheumatoid arthritis.     

Constitutive and inducible thymic stromal lymphopoietin expression in human airway smooth muscle cells: role in chronic obstructive pulmonary disease

Thymic stromal lymphopoietin (TSLP) is a novel cytokine that triggers dendritic cell-mediated T helper (Th)-2 inflammatory responses. Previous studies have demonstrated that human airway smooth muscle cells (HASMC) play a critical role in initiating or perpetuating airway inflammation by producing chemokines and cytokines. In this study, we first evaluated the expression of TSLP in primary HASMC and investigated how proinflammatory cytokines (TNF-alpha and IL-1beta) and Th-2 cytokines (IL-4, IL-9) regulate TSLP production from HASMC. TSLP mRNA and protein were assessed by real-time RT-PCR, ELISA, and immunofluorescence from primary HASMC cultures. Primary HASMC express constitutive level of TSLP. Incubation of HASMC with IL-1 or TNF-alpha resulted in a significant increase of TSLP mRNA and protein release from HASMC. Furthermore, combination of IL-1beta and TNF-alpha has an additive effect on TSLP release by HASMC. Primary HASMC pretreated with inhibitors of p38 or p42/p44 ERK MAPK, but not phosphatidylinositol 3-kinase, showed a significant decrease in TSLP release on IL-1beta and TNF-alpha treatment. Furthermore, TSLP immunoreactivity was present in ASM bundle from chronic obstructive pulmonary disease (COPD) and to lesser degree in normal subjects. Taken together, our data provide the first evidence of IL-1beta- and TNF-alpha-induced TSLP expression in HASMC via (p38, p42/p44) MAPK signaling pathways. Our results raise the possibility that HASMC may play a role in COPD airway inflammation via TSLP-dependent pathway.     

Analysis of rat testicular protein expression following 91-day exposure to JP-8 jet fuel vapor

We analyzed protein expression in preparations from whole testis in adult male Sprague-Dawley rats exposed for 6 h/d for 91 consecutive days to jet propulsion fuel-8 (JP-8) in the vapor phase (0, 250, 500, or 1000 mg/m(3) +/- 10%), simulating a range of possible human occupational exposures. Whole body inhalation exposures were carefully controlled to eliminate aerosol phase, and subjects were sacrificed within 48 h postexposure. Organ fractions were solubilized and separated via large-scale, high resolution two-dimensional electrophoresis, and gel patterns scanned, digitized and processed for statistical analysis. Seventy-six different testis proteins were significantly increased or decreased in abundance in vapor-exposed groups, compared to controls, and dose-response profiles were often nonlinear. A number of the proteins were identified by peptide mass fingerprinting and related to histopathological or physiological deficits shown in previously published studies to occur with repeated exposure to hydrocarbon fuels or solvents. These results demonstrate a significant effect of JP-8 exposure on protein expression, particularly in protein expression in the rodent testis, and suggest that a 91 d exposure to jet fuel vapor induces changes of equal or greater magnitude to those reported previously for shorter duration JP-8 aerosol exposures.     

IL-8 in septic shock, endotoxemia, and after IL-1 administration

Much effort has been directed toward elucidating the host response to sepsis and inflammation, resulting in the definition of a cascade of endogenous mediators that direct metabolic and immunological responses. Here we report that IL-8, a novel cytokine produced by a variety of cells in vitro in response to stimulation with bacterial LPS and the proinflammatory cytokines, appears in the circulation of primates in vivo during septic shock, sublethal endotoxemia, and after the administration of IL-1 alpha. The magnitude of the IL-8 response correlates with the severity of the insult, and levels of IL-8 peak relatively late, after those of TNF-alpha and IL-1 beta, and simultaneously with those of IL-6. IL-8 has been primarily defined as a selective activator and chemoattractant of neutrophils, and we demonstrate that after LPS or IL-1 alpha infusion, circulating neutrophil numbers rapidly recover from an initial neutropenia while IL-8 concentrations are maximal, supporting the hypothesis that IL-8 influences circulating leukocyte populations in vivo. We conclude that IL-8 is another participant in the cytokine cascade elicited by sepsis and inflammation and, as such, may play a significant role in host defense and disease.     

TNF-induced IL-8 and MCP-1 production in the eosinophilic cell line, EOL-1

The role of eosinophils in inflammation and their mode of activation is not well understood. Eosinophil accumulation and subsequent expression of cytokines at the site of inflammation may play a role in exacerbation of inflammatory responses. In the present study, we have examined the role of TNF-alpha in eosinophil activation and chemokine production using a human leukaemic eosinophil cell line, EOL-1. Initial studies demonstrated that TNF-alpha induced the upregulation of IL-8 and MCP-1 mRNA and protein. Kinetic studies indicated production of chemokines, IL-8 and MCP-1, as early as 4 h post-activation, with peak levels of chemokine produced at 8 h, and decreasing by 24 h post-TNF-alpha activation. When IL-10, a suppressive cytokine, was incubated with TNF-alpha and EOL-1 cells, no effect was observed on IL-8 and MCP-1 production. However, dexamethasone, a glucocorticoid, demonstrated potent inhibitory effects on the EOL-1-derived chemokines. These studies indicate that eosinophils may be a significant source of chemokines capable of participating in, and maintaining, leukocyte recruitment during inflammatory responses, such as asthma.     

Accelerated prion disease in the absence of interleukin-10

The identity of pro- and anti-inflammatory cytokines in the neuropathogenesis of prion diseases remains undefined. Here we have investigated the role of anti-inflammatory cytokines on the progression of prion disease through the use of mice that lack interleukin-4 (IL-4), IL-10, IL-13, or both IL-4 and IL-13. Collectively our data show that among these anti-inflammatory cytokines, IL-10 plays a prominent role in the regulation of prion disease. Mice deficient in IL-10 are highly susceptible to the development of prion disease and show a markedly shortened incubation time. In addition, we have correlated cytokine gene expression in prion-inoculated IL-10(-/-) mice to wild-type-inoculated animals. Our experiments show that in the absence of IL-10 there is an early expression of tumor necrosis factor alpha (TNF-alpha). In wild-type prion-inoculated mice, the expression of TNF-alpha mRNA occurs at a later time point that correlates with the extended incubation time for terminal disease development in these animals compared to those that lack IL-10. Elevated levels of IL-13 mRNA are found at early time points in the central nervous system of prion-inoculated IL-10(-/-) mice. At terminal disease, the brains of wild-type mice inoculated with RML or ME7 are characterized by elevated levels of mRNA for the proinflammatory cytokines TNF-alpha and IL-1beta, together with the anti-inflammatory cytokines IL-10, IL-13, and transforming growth factor beta. Our data are consistent with a role for proinflammatory cytokines in the initiation of pathology during prion disease and an attempt by anti-inflammatory cytokines to regulate the ensuing, invariably fatal pathology.     

Zinc-mediated inhibition of cyclic nucleotide phosphodiesterase activity and expression suppresses TNF-alpha and IL-1 beta production in monocytes by elevation of guanosine 3',5'-cyclic monophosphate

The trace element zinc affects several aspects of immune function, such as the release of proinflammatory cytokines from monocytes. We investigated the role of cyclic nucleotide signaling in zinc inhibition of LPS-induced TNF-alpha and IL-1beta release from primary human monocytes and the monocytic cell line Mono Mac1. Zinc reversibly inhibited enzyme activity of phosphodiesterase-1 (PDE-1), PDE-3, and PDE-4 in cellular lysate. It additionally reduced mRNA expression of PDE-1C, PDE-4A, and PDE-4B in intact cells. Although these PDE can also hydrolyze cAMP, only the cellular level of cGMP was increased after incubation with zinc, whereas cAMP was found to be even slightly reduced due to inhibition of its synthesis. To investigate whether an increase in cGMP alone is sufficient to inhibit cytokine release, the cGMP analogues 8-bromo-cGMP and dibutyryl cGMP as well as the NO donor S-nitrosocysteine were used. All three treatments inhibited TNF-alpha and IL-1beta release after stimulation with LPS. Inhibition of soluble guanylate cyclase-mediated cGMP synthesis with LY83583 reversed the inhibitory effect of zinc on LPS-induced cytokine release. In conclusion, inhibition of PDE by zinc abrogates the LPS-induced release of TNF-alpha and IL-1beta by increasing intracellular cGMP levels.     

Increased expression of TNF-alpha,IL-6, and IL-8 in HALS: implications for reduced adiponectin expression and plasma levels

Human immunodeficiency virus (HIV)-associated lipodystrophy syndrome (HALS) is a side effect of highly active antiretroviral therapy of HIV-infected patients; however, the mechanism of the lipodystrophy and insulin resistance seen in this syndrome remains elusive. Adiponectin, an adipocyte-specific protein, is thought to play an important role in regulating insulin sensitivity. We investigated circulating levels and gene expression of adiponectin in subcutaneous abdominal adipose tissue (AT) from 18 HIV-infected patients with HALS compared with 18 HIV-infected patients without HALS. Implications of cytokines for adiponectin levels were investigated by determining circulating levels of TNF-alpha, IL-6, and IL-8 as well as gene expression of these cytokines in AT. HALS patients exhibited 40% reduced plasma adiponectin levels (P < 0.05) compared with non-HALS subjects. Correspondingly, adiponectin mRNA levels in AT were reduced by >50% (P = 0.06). HALS patients were insulin resistant, and a positive correlation was found between plasma adiponectin and insulin sensitivity (r = 0.55, P < 0.01) and percent limb fat (r = 0.61, P < 0.01). AT mRNA of TNF-alpha, IL-6, and IL-8 was increased in AT of HALS subjects (P < 0.05), and both AT TNF-alpha mRNA and plasma TNF-alpha were negatively correlated to plasma adiponectin (P < 0.05). Finally, TNF-alpha was found in vitro to inhibit human AT adiponectin mRNA by 80% (P < 0.05). In conclusion, HALS patients have reduced levels of plasma adiponectin and adiponectin mRNA in AT. Increased cytokine mRNA in AT is hypothesized to exert an inhibitory effect on adiponectin gene expression and, consequently, to play a role in the reduced plasma adiponectin levels found in HALS patients.     

Proinflammatory cytokines inhibit human placental 11beta-hydroxysteroid dehydrogenase type 2 activity through Ca2+ and cAMP pathways

Excessive fetal exposure to glucocorticoids has been implicated in the etiology of adult metabolic and cardiovascular disease. Placental 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) may protect the fetus from excessive glucocorticoid exposure. Maternal stress may be accompanied by elevated levels of cortisol and increased proinflammatory cytokines [interleukin (IL)-1beta, IL-6, and tumor necrosis factor-alpha (TNF-alpha)]. We hypothesize that proinflammatory cytokines inhibit human placental 11beta-HSD activity. We incubated explant cultures of term human placental villi in the presence or absence of 10 ng/ml IL-1beta, IL-6, or TNF-alpha, with or without agonists or antagonists of intracellular Ca2+ and adenylyl cyclase. Activity for 11beta-HSD2 was estimated using a radioisotope assay, and mRNA was measured using quantitative RT-PCR. All cytokines significantly (P < or = 0.05) reduced 11beta-HSD2 activity (>75% suppression); maximal inhibition occurred within 2 h and was maintained for at least 24 h. The IL-1beta-induced inhibitory activity was attenuated using a Ca2+ channel blocker (nifedipine), an intracellular Ca2+ antagonist [8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate], or the adenylyl cyclase stimulant forskolin. Conversely, 11beta-HSD2 activity was diminished in the presence of the Ca2+ ionophore A-23187 or the adenylyl cyclase inhibitor SQ-22536. mRNA levels for 11beta-HSD2 were not changed by any of the treatments. Proinflammatory cytokines inhibit human placental 11beta-HSD2 activity through a mechanism that involves increased intracellular Ca2+ and inhibition of adenylyl cyclase. This could result in excessive fetal exposure to maternal cortisol. This mechanism might mediate part of the increased risk of metabolic and cardiovascular disease in adult offspring.     

Surfactant strengthens the inhibitory effect of C-reactive protein on human lung macrophage cytokine release

In this study we investigated the effect of acute-phase levels of C-reactive protein (CRP) on cytokine production by pulmonary macrophages in the presence or absence of pulmonary surfactant. Both human alveolar and interstitial macrophages as well as human surfactant were obtained from multiple organ donor lungs. Precultured macrophages were stimulated with LPS alone or together with IFN-gamma in the presence or absence of CRP, surfactant, and combinations. Releases of TNF-alpha and of IL-1beta to the medium were determined. We found that CRP could modulate lung inflammation in humans by decreasing the production of proinflammatory cytokines by both alveolar and interstitial macrophages stimulated with LPS alone or together with IFN-gamma. The potential interaction between CRP and surfactant phospholipids did not overcome the effect of either CRP or surfactant on TNF-alpha and IL-1beta release by lung macrophages. On the contrary, CRP and pulmonary surfactant together had a greater inhibitory effect than either alone on the release of proinflammatory cytokines by lung macrophages.