High expression of human monocyte iNOS mRNA induced by Paracoccidioides brasiliensis is not associated with increase in NO production

In this study we investigated the role of nitric oxide (NO) in monocyte fungicidal activity against Paracoccidioides brasiliensis. We found that cells primed with IFN-γ, TNF-α or GM-CSF and challenged with a high-(Pb18) or low-virulence (Pb265) strain of the fungus increase their fungicidal activity. Expression of iNOS mRNA was increased after priming cells with each cytokine, and tended to be inhibited by Pb18. Despite up-regulation of iNOS mRNA expression by Pb265, an equivalent increase in NO production was not detected, as metabolite levels were similar in all cultures. The results indicated that high expression of human monocyte iNOS mRNA induced by P. brasiliensis is not correlated with NO concentrations produced.     

Early response to bleomycin is characterized by different cytokine and cytokine receptor profiles in lungs

The sensitivity to the fibrosis-inducing effect of bleomycin varies considerably from species to species, the reasons for which are unknown. The variability of the response in different strains of mice is well documented. Recent evidence indicates that the upregulated expression of cytokines and cytokine receptors may be involved. We evaluated the expression pattern of some cytokines and their receptors in C57Bl/6J bleomycin-sensitive and Balb/C bleomycin-resistant mice. Animals from both strains received, under ether anesthesia, either saline (50 microl) or bleomycin (0.1 U/50 microl) intratracheally. At various times after the treatment, the lungs were analyzed for cytokines and cytokine receptors by histochemistry and their mRNA by RNase protection assay. A significantly increased expression of TNF-alpha and IL-1beta was observed in both strains. However, an upregulated lung expression for TNF-alpha and IL-1 receptors was observed in C57Bl/6J-sensitive animals only. This profile is evident from 63 h onward. In addition to TNF-alpha, bleomycin administration also resulted in the upregulated expression of TGF-beta in the lungs of both strains at 8 h and in an enhanced expression of TGF-beta receptors I and II in C57Bl/6J mice only. The upregulation of TGF-beta receptor expression was preceded in this strain by an increased expression of IL-4, IL-13, and IL-13 receptor-alpha (at 8 h after bleomycin) and followed by an upregulation of gp130 and IL-6. The difference we observed in the cytokine receptor profile may offer an additional explanation for the different fibrogenic response of the two mouse strains to bleomycin.     

Kinetics of cytokines and chemokines gene expression distinguishes Paracoccidioides brasiliensis infection from disease

The human infection with Paracoccidioides brasiliensis may result in three major outcomes: the paracoccidioidomycosis-infection (PI), the adult form (AF) and the juvenile form (JF) of the disease. The aim of this study was to compare the immunological response among these groups. The gene expression of multiple cytokines, including IL-4, IL-5, IL-6, IL-10, IFN-gamma, TNF-alpha and TGF-beta1, and chemokines, CXCL8, CXCL9 and CXCL10 was evaluated by RT-PCR in peripheral blood mononuclear cells unstimulated or following phytohemagglutinin stimulation for 3, 6, 12, 24 and 48 h. PI individuals expressed earlier and higher levels of mRNA of IFN-gamma, TNF-alpha, CXCL9 and CXCL10 when compared to JF patients. In relation to AF patients, the PI group presented similar levels of CXCL10 and IFN-gamma and higher levels of CXCL9. On the other hand, mRNA expression of Th2 cytokines (IL-4, IL-10, IL-5 and TGF-beta1) was higher and earlier in JF and AF groups, when compared to PI individuals. At some time intervals it was possible to differentiate JF from AF, mainly in relation to IL-4 and TGF-beta1 mRNA, expressed in higher levels in the JF patients. The distinct patterns of cytokines and chemokines expression support their important role in determining the different outcomes observed in this disease.     

Human first trimester forebrain cells express genes for inflammatory and anti-inflammatory cytokines

Using in situ hybridization and immunohistochemistry the induction of potential cytokines [interleukin 1beta (IL-1beta), IL-4, IL-6, IL-10, tumour necrosis factor alpha (TNF-alpha), interferon gamma (IFN-gamma) and transforming growth factor beta (TGF-beta)] in human embryonic forebrain cells at weeks 5, 7 and 10 of gestation was studied. The aims were to investigate the capacity of these cells to express cytokines, to determine the kinetics of induction and to display the type of cytokine expressing cells in the developing brain. Constitutive cytokine gene expression was recorded from week 5, augmented by about 50% at week 7 and by more than 100% at week 19 (except TGF-beta at week 10). Among other cytokines, IL-1beta exhibited the highest expression at week 10. TGF-beta showed maximum expression at week 7 and declined at week 10. Combined techniques revealed that glial cells are a major source of cytokines. The study show and present for the first time the kinetics of spontaneous cytokine expression in human embryonic forebrain cells. The increased mRNA expressions with age suggest an important role for cytokines in promotion of brain development. The capacity of inducing these cytokines may, however, be implicated in the immunopathogenesis of several brain diseases. The distinctive TGF-beta profile suggests a further role for TGF-beta on modulation of cytokine responses during development.     

Systemic cytokine response in murine anthrax

Systemic pro-inflammatory cytokine release has been previously implicated as a major death-causing factor in anthrax, however, direct data have been absent. We determined the levels of IL-1 beta, IL-6 and TNF-alpha in serum of mice challenged with virulent (Ames) or attenuated (Sterne) strains of Bacillus anthracis. More than 10-fold increase in the IL-1beta levels was detected in Ames-challenged Balb/c mice, in contrast to more susceptible C57BL/6 mice, which showed no IL-1beta response. Balb/c mice have also responded with higher levels of IL-6. The A/J mice demonstrated IL-1beta and IL-6 systemic response to either Ames or Sterne strain of B. anthracis, whereas no increase in TNF-alpha was detected in any murine strain. We used RT-PCR for gene expression analyses in the liver which often is a major source of cytokines and one of the main targets in infectious diseases. A/J mice challenged with B. anthracis (Sterne) showed increased gene expression for Fas, FasL, Bax, IL-1 beta, TNF-alpha, TGF-beta, MIP-1alpha, KC and RANTES. These data favour the hypothesis that apoptotic cell death during anthrax infection causes chemokine-induced transmigration of inflammatory cells to vitally important organs such as liver. Administration of caspase inhibitors z-VAD-fmk and ac-YVAD-cmk improved survival in Sterne-challenged mice indicating a pathogenic role of apoptosis in anthrax.     

Regulation of rat pulmonary artery endothelial cell transforming growth factor-beta production by IL-1 beta and tumor necrosis factor-alpha.

Recent studies suggest that transforming growth factor-beta (TGF-beta) production is up-regulated at sites of tissue injury, inflammation and repair, or fibrosis. Endothelial cells represent a potentially important in vivo source of TGF-beta; however, the identity of endogenous modulators of TGF-beta production by these cells remains unclear. To address this issue, the effects of the cytokines, IL-1 beta, and TNF-alpha on TGF-beta production by rat pulmonary artery endothelial cells were examined. Conditioned media from cells treated with 0 to 20 ng/ml IL-1 beta and/or TNF-alpha were assayed for TGF-beta activity using a mink lung epithelial cell line. The results show that rat pulmonary artery endothelial cells secreted undetectable amounts of active TGF-beta in the absence of cytokines. However, upon acidification of the conditioned media before assay, a time-dependent increase in TGF-beta activity was noted in media from both untreated and cytokine-treated cells. However, both IL-1 beta and TNF-alpha treatment caused the secretion of significantly greater amounts of TGF-beta activity than control cells, in a dose-dependent manner, with maximal response obtained at cytokine doses of greater than 10 ng/ml. At equivalent doses of cytokine tested, the magnitude of the response was significantly greater with IL-1 beta. These responses were paralleled by increases in steady state mRNA levels for TGF-beta 1. Addition of both cytokines resulted in a synergistic response. Synergism with IL-1 beta was also noted with the fibrogenic agent bleomycin. Kinetic studies indicated that a minimum of 4 h of treatment with either IL-1 beta or TNF-alpha was required for detection of significant increases in either secreted TGF-beta activity or steady state TGF-beta 1 mRNA levels. Thus, endothelial cells could play a role in various TGF-beta-dependent processes in vivo, in situations wherein IL-1 beta and/or TNF-alpha may be present at comparable concentrations.     

The effect of interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) on granulocyte macrophage-colony stimulating factor (GM-CSF) production by neuronal precursor cells

AIMS: To determine whether granulocyte macrophage-colony stimulating factor (GM-CSF) production by neuronal precursor (NT2) cells can be regulated by IL-1beta and TNF-alpha. BACKGROUND: We have previously demonstrated GM-CSF expression by neurons of the developing human brain, as well as by NT2 cells. IL-1beta and TNF-alpha upregulate GM-CSF production in glial cells, but GM-CSF regulation in neurons is as yet undefined. We hypothesized that IL-1beta and TNF-alpha would increase GM-CSF mRNA and protein production in NT2 cells. METHODS: The effect of IL-1beta and TNF-alpha on GM-CSF production was assessed by dose response (0 to 2,000 U/ml), and time course (0 to 48 hours incubation) experiments. GM-CSF mRNA and protein production were assessed by quantitative RT-PCR and by ELISA. The effect of these cytokines on cell turnover was determined by BrdU incorporation. RESULTS: IL-1beta increased GM-CSF mRNA and protein expression by NT2 cells. This effect was time and dose dependent, and the effective dose ranging from (20-200 U/ml). TNF-alpha increased GM-CSF mRNA expression to a lesser extent than did IL-1beta (maximal stimulation at 200 U/ml), and a minimal increase in net protein accumulation was noted. Neither cytokine increased NT2 cell turnover. CONCLUSIONS: IL-1beta and TNF-alpha both increase GM-CSF mRNA expression by NT2 cells, but only IL-1beta increases net GM-CSF protein accumulation.     

Expression of cytokines by multipotent neural progenitor cells

Recent work with mammalian neural stem cells has highlighted the role of cytokine signaling in the proliferation and differentiation of these multipotent cells. While the responsiveness of neural progenitors to exogenously applied growth factors has been demonstrated in vivo as well as in vitro, little attention has been given to the production of cytokines by these cells. Here we use immunocytochemistry, RT-PCR, and ELISA to show that under standard growth conditions multipotent neural progenitor cells from humans express multiple cytokines including IL-1alpha, IL-1beta, IL-6, TGF-beta1, TGF-beta2, TNF-alpha, but not IL-2, IL-4, or IFN-gamma. Neural progenitor cells from rat and mouse express some, but not all, of these cytokines under similar conditions. While the function of cytokine expression by neural progenitor cells remains to be elucidated, these signaling molecules are known to be involved in neural development and may play a role in the activation of quiescent stem cells by a variety of pathological processes.     

Systemic and local cytokine response of young piglets to oral infection with<Emphasis Type="Italic">Salmonella enterica</Emphasis> serotype typhimurium

One-week-old breast-fed miniature piglets were orally infected either with virulent LT2 strain or with a non-virulent SF1591 rough mutant of Salmonella Typhimurium for 1 d. Both microorganisms were cultivated from mesenteric lymph nodes but not from the blood of infected piglets. Interleukins (IL) 1 beta, 8, 18, tumor necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma) were quantified by ELISA in plasma and washes of a terminal part of the small bowel. In plasma, cytokines were mostly missing in non-infected piglets and either missing or low in infected piglets. In the gut of non-infected piglets, IL-1 beta, IL-8 and IL-18 were detected whereas TNF-alpha and IFN-gamma were mostly missing. IFN-gamma levels highly increased (p < 0.05) after infection with nonvirulent salmonellae. The variability of cytokine levels in the gut of suckling piglets is discussed.     

Cytokine gene expression--part of host defence in pulpitis

Analyses of cytokines mediating inflammatory reactions are key to understanding the etiopathology of various diseases. This study investigated differences in cytokine gene expression between pulps from healthy virgin teeth and from symptomatic vital teeth with severe caries lesions in a group of young, healthy individuals. The mRNA levels of IL-1alpha, IL-1beta, IL-6, IL-8, and IL-18 were measured concomitantly by quantitative real-time RT-PCR. IL-1alpha and IL-1beta were not expressed at significantly higher levels in symptomatic versus clinically healthy pulps, while the difference was significant for the other cytokines (log-rank test, P<0.05). A concordance test for independence revealed significant correlation between IL-1alpha and IL-1beta, and between IL-6, IL-8, and IL-18 mRNA levels (P<0.05). The cytokine-specific differences revealed a differential significance of gene expression in cytokine regulation. The hypothesis that increase of cytokine mRNA expression is part of host reaction in pulpitis was corroborated by our observation.     

The transcriptional control of TGF-beta in human osteoblast-like cells is distinct from that of IL-1 beta.

Transforming growth factor beta (TGF-beta) and interleukin 1 (IL-1) are among the most potent osteotropic cytokines. The expression of mRNA for both TGF-beta and IL-1 beta was studied in human osteoblast-like cells in vitro. These cells constitutively expressed TGF-beta but not IL-1 beta mRNA. Treatment of the cells with the systemic hormones 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] (10(-8) M) and parathyroid hormone (10(-7) M) induced an increase in TGF-beta mRNA but failed to stimulate the production of IL-1-beta mRNA. Retinoic acid (10(-8) M) had no effect on either mRNA species. The cytokines IL-1 alpha (200 pg/ml), tumour necrosis factor alpha (TNF-alpha) (17 ng/ml) and bacterial lipopolysaccharide (LPS) (500 ng/ml) stimulated the production of IL-1 beta mRNA after 6-8 hours. This was followed by an increase in protein production after 24 hours. In contrast, the production of TGF-beta mRNA remained constant after treatment with these agents. Treatment of the cells with hydrocortisone (10(-8) M) resulted in the suppression of both TGF-beta and IL-1 beta mRNA. However, when the stimulating agent 1,25-(OH)2D3 was added in conjunction with hydrocortisone the mRNA expression of TGF-beta mRNA returned to 70% of the stimulated level. In contrast, the addition of the stimulatory agent IL-1 alpha to hydrocortisone-treated cells resulted in no increase in IL-1 beta mRNA. In-situ hybridization demonstrated both TGF-beta and IL-1 beta mRNA at the cellular level.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of cytokines on the in vitro fungicidal activity of monocytes from paracoccidioidomycosis patients

Peripheral blood monocytes obtained from paracoccidioidomycosis patients and healthy individuals were preactivated with recombinant gamma interferon (IFN-gamma) in different concentrations (250, 500 and 1000 U/ml) and evaluated for fungicidal activity against Paracoccidiodes brasiliensis strain 18 (Pb 18, high-virulence strain) and strain 265 (Pb 265, low-virulence strain) by plating of cocultures and counting of colony-forming units, after 10 d. Monocytes from healthy individuals failed to present fungicidal activity against P. brasiliensis even after IFN-gamma activation at the three concentrations. However, patient monocytes activated with IFN-gamma (1000 U/ml) showed a significant fungicidal activity when compared to that obtained with non-activated or activated cells with other IFN-gamma concentrations (250 and 500 U/ml). Moreover, patient monocytes presented higher fungicidal activity than the control, even before the activation process. These results may be explained by the activation state of patients' cells as a function of the in vivo contact with the fungus, which was confirmed by their higher capacity to release H(2)O(2) in vitro. Unlike the results obtained with Pb 18, patient and control cells presented a significant fungicidal activity against Pb 265, after priming with IFN- gamma. These results are explained by the higher levels of TNF-alpha in supernatants of cultures challenged with Pb 265. Moreover, higher levels of the cytokine were obtained in patient cell supernatants. Taken together, our results suggest that for effective killing of P. brasiliensis by monocytes, an initial activation signal induced by IFN-gamma is necessary to stimulate the cells to produce TNF-alpha. This cytokine may be involved, through an autocrine pathway, in the final phase activation process. The effectiveness of this process seems to depend on the virulence of the fungal strain and the activation state of the challenged cells.     

Quercetin's influence on exercise-induced changes in plasma cytokines and muscle and leukocyte cytokine mRNA.

Trained male cyclists (n = 40) ingested quercetin (Q; n = 20) (1,000 mg/day) or placebo (P; n = 20) supplements under randomized, double-blinded methods for 3 wk before and during a 3-day period in which subjects cycled for 3 h/day at approximately 57% maximal work rate. Blood samples were collected before and after each exercise session and assayed for plasma IL-6, IL-10, IL-1ra, IL-8, TNF-alpha, and monocyte chemoattractant protein 1, and leukocyte IL-10, IL-8, and IL-1ra mRNA. Muscle biopsies were obtained before and after the first and third exercise sessions and assayed for NF-kappaB and cyclooxygenase-2 (COX-2), IL-6, IL-8, IL-1beta, and TNF-alpha mRNA. Postexercise increases in plasma cytokines did not differ between groups, but the pattern of change over the 3-day exercise period tended to be lower in Q vs. P for IL-8 and TNF-alpha (P = 0.094 for both). mRNA increased significantly postexercise for each cytokine measured in blood leukocyte and muscle samples. Leukocyte IL-8 and IL-10 mRNA were significantly reduced in Q vs. P (interaction effects, P = 0.019 and 0.012, respectively) with no other leukocyte or muscle mRNA group differences. Muscle NF-kappaB did not increase postexercise and did not differ between Q and P. Muscle COX-2 mRNA increased significantly postexercise but did not differ between Q and P. In summary, 1 g/day quercetin supplementation by trained cyclists over a 24-day period diminished postexercise expression of leukocyte IL-8 and IL-10 mRNA, indicating that elevated plasma quercetin levels exerted some effects within the blood compartment. Quercetin did not, however, influence any of the muscle measures, including NF-kappaB content, cytokine mRNA, or COX-2 mRNA expression across a 3-day intensified exercise period.     

Antioxidants attenuate the plasma cytokine response to exercise in humans.

Exercise increases plasma TNF-alpha, IL-1beta, and IL-6, yet the stimuli and sources of TNF-alpha and IL-1beta remain largely unknown. We tested the role of oxidative stress and the potential contribution of monocytes in this cytokine (especially IL-1beta) response in previously untrained individuals. Six healthy nonathletes performed two 45-min bicycle exercise sessions at 70% of Vo(2 max) before and after a combination of antioxidants (vitamins E, A, and C for 60 days; allopurinol for 15 days; and N-acetylcysteine for 3 days). Blood was drawn at baseline, end-exercise, and 30 and 120 min postexercise. Plasma cytokines were determined by ELISA and monocyte intracellular cytokine level by flow cytometry. Before antioxidants, TNF-alpha increased by 60%, IL-1beta by threefold, and IL-6 by sixfold secondary to exercise (P < 0.05). After antioxidants, plasma IL-1beta became undetectable, the TNF-alpha response to exercise was abolished, and the IL-6 response was significantly blunted (P < 0.05). Exercise did not increase the percentage of monocytes producing the cytokines or their mean fluorescence intensity. We conclude that in untrained humans oxidative stress is a major stimulus for exercise-induced cytokine production and that monocytes play no role in this process.