Production Of Reactive Nitrogen Intermediates Research Articles

Effect of oestrogen on Mycobacterium avium complex pulmonary infection in mice.

The purpose of the present study was to elucidate the role of oestrogen in the pathogenesis of Mycobacterium avium complex (MAC) pulmonary disease, which occurs most frequently in postmenopausal women. The study was carried out in a murine infectious model using ovariectomized DBA/2 female mice. Infection with MAC was established by intratracheal administration of bacilli. In some experiments, ovariectomized mice were treated with exogenous 17 beta-estradiol (E2). The number of bacilli in the lungs of infected mice which received ovariectomy was significantly larger than that in the lungs of sham-operated control mice, and treatment of ovariectomized mice with exogenous E2 restored the burden of bacilli to the same level as that in the sham-operated control mice. We next examined the effect of E2 in vitro using bone marrow-derived macrophages obtained from DBA/2 female mice. The macrophages showed bacteriostatic activity against MAC after treatment with interferon-gamma (IFN-gamma) and this activity was further enhanced by the exogenous addition of E2 to the culture medium. In parallel with these findings, E2 augmented the production of reactive nitrogen intermediates (RNI) by macrophages pretreated with IFN-gamma and stimulated with MAC, as shown by evaluating nitrite production and inducible nitric oxide synthase mRNA expression. These findings taken together suggest that absence of endogenous oestrogen appears to be responsible for the development of MAC pulmonary disease in this mouse model and that the enhancement by E2 of anti-MAC activity of murine macrophages induced through increased RNI production may play some role in resistance to MAC infection.

Influence of macrophage resistance gene Lsh/Ity/Bcg (candidate Nramp) on Toxoplasma gondii infection in mice.

Functional studies have shown that the murine macrophage resistance gene Lsh/Ity/Bcg (candidate Nramp) regulates macrophage priming/activation for antimicrobial activity via the tumour necrosis factor-alpha (TNF-alpha)-dependent production of reactive nitrogen intermediates. Since Toxoplasma gondii also parasitizes macrophages, is a stimulator of endogenous TNF-alpha release, and is sensitive to nitric oxide-mediated killing in activated macrophages, studies were carried out using chromosome 1 congenic mouse strains to determine whether Lsh influences T. gondii infection. Two interesting observations were made: (i) contrary to expectation, mice carrying the Lsh-resistant allele died earlier over the acute phase of infection than Lsh-susceptible mice; and (ii) Lsh-resistant mice which survived this acute phase of infection showed lower brain cyst numbers than the Lsh-susceptible mice. Whilst the latter occurred independently of route of inoculation (oral, intraperitoneal, or subcutaneous), the former was influenced both by the route of inoculation and the genetic background on which the Lsh-resistant allele had been isolated. Hence, following oral administration of 20 brain cysts of the RRA strain of T. gondii, mice carrying the Lsh-resistant allele on a B10 genetic background showed a significantly enhanced rate of mortality over the acute (first 8-12 days) phase of infection than B10 Lsh-susceptible mice. Although this acute phase of infection in B10 background mice was accompanied by an increase in serum TNF-alpha levels in both Lsh-resistant and -susceptible mouse strains, early mortality preceded the TNF-alpha peak, and administration of neutralizing rabbit anti-TNF-alpha did not significantly enhance survival. Hence, inflammatory mediators other than TNF-alpha appear to be responsible for the increased rate of acute mortality observed in resistant mice. Infection intraperitoneally led to delayed mortality in B10 mice, with the mean time to 50% mortality now being significantly longer in Lsh-resistant than in Lsh-susceptible mice. On a BALB genetic background, it was the i.p. route of infection which led to acute mortality and more rapid death in the Lsh-resistant strain. When a less virulent inoculum was used and mortality delayed, Lsh-susceptible mice died more rapidly, and i.p. administration of rabbit anti-TNF-alpha led to 100% mortality between days 8 and 10 of infection in both susceptible and resistant mouse strains, consistent with a crucial protective role for TNF-alpha during this phase of infection.(ABSTRACT TRUNCATED AT 400 WORDS)

Production of reactive nitrogen intermediates by bone marrow-derived macrophages on treatment with cisplatin in vitro.

L929 culture medium (a source of macrophage colony stimulating factor (M-CSF) or recombinant granulocyte-macrophage colony stimulating factor (rGM-CSF)-derived bone marrow macrophages treated with cisplatin or lipopolysaccharide (LPS) (10 micrograms/ml) were effective in the production of L-arginine-dependent reactive nitrogen intermediates (RNI) and generation of tumouricidal activity. The abilities of RNI secretion and related tumouricidal activity against P815 mastocytoma cells were compared. These parameters were found to be closely correlated in various experiments. RNI secretion and generation of bone marrow macrophage-mediated tumouricidal activity were significantly inhibited by L-N-monomethyl arginine (L-NMMA), a specific inhibitor of the L-arginine pathway, but L-NMMA did not inhibit macrophage-mediated killing of tumour necrosis factor (TNF)-sensitive Wehi cells, suggesting that activated macrophages exhibit at least two cytolytic mechanisms, one by L-arginine-dependent nitric oxide pathway and another by TNF-mediated killing. The present findings suggest that the mechanism of tumour cell killing by activated macrophages may differ, depending on the tumour cell type, and reactive nitrogen intermediates play a major role in cisplatin-mediated activation of bone marrow-derived macrophages.

Detrimental role of endogenous nitric oxide in host defence against Sporothrix schenckii

We earlier demonstrated that nitric oxide (NO) is a fungicidal molecule against Sporothrix schenckii in vitro. In the present study we used mice deficient in inducible nitric oxide synthase (iNOS-/-) and C57BL/6 wild-type (WT) mice treated with Nomega-nitro-arginine (Nitro-Arg-treated mice), an NOS inhibitor, both defective in the production of reactive nitrogen intermediates, to investigate the role of endogenous NO during systemic sporotrichosis. When inoculated with yeast cells of S. schenckii, WT mice presented T-cell suppression and high tissue fungal dissemination, succumbing to infection. Furthermore, susceptibility of mice seems to be related to apoptosis and high interleukin-10 and tumour necrosis factor-alpha production by spleen cells. In addition, fungicidal activity and NO production by interferon-gamma (IFN-gamma) and lipopolysaccharide-activated macrophages from WT mice were abolished after fungal infection. Strikingly, iNOS-/- and Nitro-Arg-treated mice presented fungal resistance, controlling fungal load in tissues and restoring T-cell activity, as well as producing high amounts of IFN-gamma Interestingly, macrophages from these groups of mice presented fungicidal activity after in vitro stimulation with higher doses of IFN-gamma. Herein, these results suggest that although NO was an essential mediator to the in vitro killing of S. schenckii by macrophages, the activation of NO system in vivo contributes to the immunosuppression and cytokine balance during early phases of infection with S. schenckii.

Epigallocatechin gallate inhibits intracellular survival of Listeria monocytogenes in macrophages

Epigallocatechin gallate (EGCg), the major tea catechin, is known as a potent anti-microbial and anti-tumor compound. The effects of EGCg on host defense mechanisms against Listeria monocytogenes infection were examined in vitro using mouse peritoneal exudate cells. The study showed that EGCg inhibited the intracellular growth of L. monocytogenes in macrophages. The enhancement of in vitro anti- L. monocytogenes activity by EGCg is not due to the modulation of reactive oxygen intermediates or the production of reactive nitrogen intermediates but due to the inhibition of its escaping from the phagosome into cytosolic space. Anti- L. monocytogenes of EGCg is through the inhibition of hemolytic and cholesterol-binding activity of listeriolysin O, which usually disrupts the phagosomal membrane in the escaping phase of L. monocytogenes.

Vav proteins control MyD88-dependent oxidative burst

AbstractThe importance of reactive oxygen intermediate (ROI) production in antimicrobial responses is demonstrated in human patients who suffer from chronic granulomatous disease (CGD) due to defective NADPH oxidase function. Exactly how bacterial products activating Toll-like receptors (TLRs) induce oxidative burst is unknown. Here, we identify the Vav family of Rho guanine nucleotide exchange factors (GEFs) as critical mediators of LPS-induced MyD88-dependent activation of Rac2, NADPH oxidase, and ROI production using mice deficient in Vav1, Vav2, and Vav3. Vav proteins are also required for p38 MAPK activation and for normal regulation of proinflammatory cytokine production, but not for other MyD88-controlled effector pathways such as those involving JNK, COX2, or iNOS and the production of reactive nitrogen intermediates (RNIs). Thus, our data indicate that Vav specifically transduces a subset of signals emanating from MyD88.

Immune response in the tilapia, Oreochromis mossambicus on exposure to tannery effluent

The objective of the study was to investigate the effect of chronic exposure to sublethal concentrations of tannery effluent (TE) on the specific immune response and nonspecific immunity in tilapia, Oreochromis mossambicus. The effluent from the tannery was collected directly from a chrome-tanning factory situated in Dindigul district, Tamil Nadu, India. Apart from chromium (88.2 ppm), the effluent contained appreciable amount of calcium carbonate and sodium sulphate. Groups of fish (45–50 g) were exposed to 0.0053, 0.053 or 0.53% [0.1%, 1% or 10% LC 50] of TE for 28 days. The specific immune response of fish was assessed by antibody response to heat-killed Aeromonas hydrophila by ELISA and bacterial agglutination assay. Nonspecific immune mechanisms were assessed in terms of serum lysozyme activity, production of intracellular reactive oxygen species (ROS) and reactive nitrogen intermediates (RNI) by peripheral blood leucocytes (PBL). The results indicate that chronic exposure of fish to 0.53% of TE, significantly suppressed antibody response, nonspecific serum lysozyme activity, and ROS and RNI production. Exposure to 0.053% (1% LC 50) of TE also caused a similar suppressive effect though at a lesser degree. In conclusion, the study shows, that exposure to sublethal concentrations of TE, can lead to adverse effects on selected immune reactions in tilapia. Further, these findings may be important in terms of monitoring fish health and risk assessment during periods of fluctuating levels of pollutants in the natural and farm environments.

Measurement and Significance of 3‐Nitrotyrosine in Systemic Lupus Erythematosus

Nitration of free and protein associated tyrosine represents, in vivo, a mechanism that can severely compromise the cell function. The detection of 3-nitrotyrosine (3-NT) in pathological tissues is suggestive of the occurrence of nitrating pathways and has been identified as a marker of inflammation and a stable end product of increased reactive nitrogen intermediate production. Protein nitration occurs in many disease conditions including systemic lupus erythematosus (SLE). In this study we show that the level of both free and protein bound 3-NT, which is produced by reactive nitrogen species (RNS)-dependent oxidative damage, is elevated in patients with SLE and that there is a possible role of RNS-modified epitopes in the aetiology of the disease. Commercially available poly L-tyrosine was exposed to nitrating species, inducing nitration in tyrosine residues. Immunoglobulin-G (IgG) purified on Protein-A-Sepharose matrix from 24 SLE patients was studied for their recognition of native and nitrated poly L-tyrosine by direct binding and competition enzyme-linked immunosorbent assay (ELISA). The formation of immune complex between SLE IgG and nitrated poly L-tyrosine was visualized by gel retardation assay. Free 3-NT in patients' sera was detected and quantitated by high performance liquid chromatography whereas protein-bound 3-NT was analysed by Western blotting and the concentration was calculated by sandwich ELISA. The concentration of free 3-NT was found to be 1.4 +/- 0.09 microm whereas the concentration of protein bound 3-NT was 96.52 +/- 21.12 microm nitrated bovine serum albumin equivalents/mg protein, which was significantly higher when compared with healthy controls. Elevated level of 3-NT was observed in SLE patients using two different techniques, when compared with healthy subjects confirms the overproduction of RNS in the pathogenesis of human SLE.

TGF receptor II gene deletion in leucocytes prevents cerebral vasculitis in bacterial meningitis

In bacterial meningitis, chemokines lead to recruitment of polymorphonuclear leucocytes (PMN) into the CNS. At the site of infection in the subarachnoid space, PMN release reactive oxygen species, reactive nitrogen intermediates (RNI) and interleukin-1beta (IL-1beta). Although these immune factors assist in clearance of bacteria, they also result in neuronal injury associated with meningitis. Transforming growth factor beta (TGFbeta) is a potent deactivator of PMN and macrophages since TGFbeta suppresses the production of ROI, RNI and IL-1. Here, we report that the deletion of the TGFbeta receptor II gene in PMN enhances PMN recruitment into the CNS of mice with Streptococcus pneumoniae meningitis. This was associated with more efficient clearance of bacteria, and almost complete prevention of intracerebral necrotizing vasculitis. Differences in PMN in the CNS of infected control mice and mice lacking TGFbeta receptor II were not explained by altered expression of chemokines acting on PMN. Instead, TGFbeta was found to impair the expression of L (leucocyte)-selectin on PMN from control mice but not from mice lacking TGFbeta receptor II. L-selectin is known to be essential for PMN recruitment in bacterial meningitis. We conclude that defective TGFbeta signalling in PMN is beneficial in bacterial meningitis by ameliorating migration of PMN and bacterial clearance.

Anti-inflammatory effect of augmented nitric oxide production in chronic lung infection

Chronic infection of the lungs with Pseudomonas aeruginosa complicates many long-term lung diseases including cystic fibrosis, bronchiectasis, chronic obstructive lung disease, and mechanical ventilation. In acute inflammatory lung diseases, increased nitric oxide synthase (NOS-2) expression leads to excess nitric oxide (NO) production, resulting in the production of reactive nitrogen intermediates, which contribute to tissue damage. In contrast, the contribution of NO to pulmonary damage in chronic Pseudomonas infection of the lung has not been directly examined and is unclear. Although NOS-2 expression is increased in this condition, NO production is not abnormally elevated. It was hypothesized that chronic infection of the airways does not cause increased NO production but, in contrast, leads to inappropriately low NO concentrations that are pro-inflammatory. A rodent model of chronic airway infection was used to examine the effects on lung damage of augmenting or inhibiting NO production after airway infection with P. aeruginosa was well established. Three days post-infection, L-arginine, which augments NO production, or L-NAME, an inhibitor of NO production, was administered in drinking water. Lung damage was assessed 12 days later. L-arginine treatment reduced tissue damage, inhibited neutrophil recruitment, and reduced the pro-inflammatory cytokine interleukin (IL)-1beta. Treatment with L-NAME caused loss of alveolar walls, greater vascular damage, and increased levels of the pro-inflammatory cytokine IL-6. Thus, in chronic airway infection, inhibition of NO production worsened lung damage, whereas augmenting NO ameliorated this damage. This is the first demonstration that augmenting endogenous NO production in chronic infective lung disease caused by P. aeruginosa is anti-inflammatory. Given that infection with this organism complicates many chronic lung diseases, most notoriously cystic fibrosis, these findings have important clinical implications.

Absence of complement receptor 3 results in reduced binding and ingestion of Mycobacterium tuberculosis but has no significant effect on the induction of reactive oxygen and nitrogen intermediates or on the survival of the bacteria in resident and interferon-gamma activated macrophages

The interaction of host macrophage (Mϕ) and Mycobacterium tuberculosis (Mtb) is mediated by cell surface receptors and is important in establishing intracellular infection. Mϕs can kill invading organisms via reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI). Using a Complement Receptor 3 (CR3) knockout mouse model we have examined whether the presence of CR3 affects the binding and uptake of viable Mtb by Mϕs, the survival of the ingested bacteria and the induction of ROI and RNI during this interaction. We show that, although CR3 plays a role in the uptake of viable Mtb, the receptor plays no role in the subsequent survival of the bacteria. The finding holds true for resident Mϕs and for interferon-gamma (IFN-γ) activated Mϕs, both in the absence and presence of serum opsonins. Activation of Mϕ populations with IFN-γ significantly inhibits the growth of Mtb in host Mϕs and enhances the production of ROI and RNI. However, the presence of CR3 was not critical in any of these mechanisms. Furthermore, we demonstrate that the control of intracellular growth of Mtb in IFN-γ activated Mϕs is not mediated by a direct effect of RNI.

CD40 Signaling in Macrophages Induces Activity against an Intracellular Pathogen Independently of Gamma Interferon and Reactive Nitrogen Intermediates

Gamma interferon (IFN-gamma) is the major inducer of classical activation of macrophages. Classically activated mouse macrophages acquire antimicrobial activity that is largely dependent on the production of reactive nitrogen intermediates. However, protection against important intracellular pathogens can take place in the absence of IFN-gamma and nitric oxide synthase 2 (NOS2). Using Toxoplasma gondii as a model, we investigated if CD40 signaling generates mouse macrophages with effector function against an intracellular pathogen despite the absence of priming with IFN-gamma and lack of production of reactive nitrogen intermediates. CD40-stimulated macrophages acquired anti-T. gondii activity that was not inhibited by a neutralizing anti-IFN-gamma monoclonal antibody but was ablated by the neutralization of tumor necrosis factor alpha (TNF-alpha). Moreover, while the induction of anti-T. gondii activity in response to CD40 stimulation was unimpaired in macrophages from IFN-gamma(-/-) mice, macrophages from TNF receptor 1/2(-/-) mice failed to respond to CD40 engagement. In contrast to IFN-gamma-lipopolysaccharide, CD40 stimulation did not induce NOS2 expression and did not trigger production of reactive nitrogen intermediates. Neither N(G)-monomethyl-l-arginine nor diphenyleneiodonium chloride affected the induction of anti-T. gondii activity in response to CD40. Finally, macrophages from NOS2(-/-) mice acquired anti-T. gondii activity in response to CD40 stimulation that was similar to that of macrophages from wild-type mice. These results demonstrate that CD40 induces the antimicrobial activity of macrophages against an intracellular pathogen despite the lack of two central features of classically activated macrophages: priming with IFN-gamma and production of reactive nitrogen intermediates.

Legionella pneumophila Infection Possibly Related to Treatment With Infliximab

Legionella pneumophila Infection Possibly Related to Treatment With Infliximab

Reactive nitrogen intermediates in experimental trichomoniasis induced with isolates from symptomatic and asymptomatic women.

Trichomoniasis is the interplay between the infecting parasite, Trichomonas vaginalis, and the host, on which the clinical presentation of the disease depends. Although the clinical spectrum varies from an asymptomatic state to mild, moderate or severe symptoms, the exact virulence markers of T. vaginalis have not been well elucidated. Free radical generation during the disease process and its role in pathogenesis has been reported in various microbial diseases. In the present study, an attempt has been made to study reactive nitrogen intermediate (RNI) concentrations in experimental animals infected with T. vaginalis isolates from symptomatic and asymptomatic women. A significant increase in polymorphs, vaginal epithelial cells and RNI levels was observed in mice infected with isolates from symptomatic and asymptomatic subjects as compared to uninfected controls. The mean concentration of RNI in the vaginal tissue of mice infected with isolates from symptomatic women (75.5+/-7.7) was significantly higher than that of the vaginal tissue of mice infected with isolates from asymptomatic women (47.9+/-7.8), while it was less in the vaginal washes and plasma of mice infected with isolates from symptomatic women (18.7+/-3.6 and 17.1+/-3.3, respectively) compared to those infected with isolates from asymptomatic women (28.9+/-7.3 and 26.7+/-4.4, respectively), which may be due to different macrophage populations with different functional capabilities. Our study indicates that RNI production may play a role in establishing the infection.

Intercellular adhesion molecule 1 deficiency leads to impaired recruitment of T lymphocytes and enhanced host susceptibility to infection with Trypanosoma cruzi.

In this study, we investigated the involvement of Th1 cytokines in the expression of cell adhesion molecules (CAM) and recruitment of inflammatory cells to the heart of mice infected with Trypanosoma cruzi. Our results show that endogenously produced IFN-gamma is essential to induce optimal expression of VCAM-1 and ICAM-1 on the cardiac vascular endothelium of infected mice. Furthermore, the influx of inflammatory cells into the cardiac tissue was impaired in Th1 cytokine-deficient infected mice, paralleling the intensity of VCAM-1 and ICAM-1 expression on the vascular endothelium. Consistent with the importance of ICAM-1 in host resistance, ICAM-1 knockout (KO) mice were highly susceptible to T. cruzi infection, as assessed by mortality rate, parasitemia, and heart tissue parasitism. The enhanced parasitism was associated with a decrease in the numbers of CD4(+) and CD8(+) T lymphocytes in the heart tissue of ICAM-1 KO mice. Additionally, ICAM-1 KO mice mounted an unimpaired IFN-gamma response and IFN-gamma-dependent production of reactive nitrogen intermediates and parasite- specific IgG2a. Supporting the participation of ICAM-1 in cell migration during T. cruzi infection, the entrance of adoptively transferred PBL from T. cruzi-infected wild-type C57BL/6 mice into the cardiac tissue of ICAM-1 KO mice was significantly abrogated. Therefore, we favor the hypothesis that ICAM-1 plays a crucial role in T lymphocyte recruitment to the cardiac tissue and host susceptibility during T. cruzi infection.

Involvement of the intracellular ion channel CLIC1 in microglia-mediated beta-amyloid-induced neurotoxicity.

It is widely believed that the inflammatory events mediated by microglial activation contribute to several neurodegenerative processes. Alzheimer's disease, for example, is characterized by an accumulation of beta-amyloid protein (Abeta) in neuritic plaques that are infiltrated by reactive microglia and astrocytes. Although Abeta and its fragment 25-35 exert a direct toxic effect on neurons, they also activate microglia. Microglial activation is accompanied by morphological changes, cell proliferation, and release of various cytokines and growth factors. A number of scientific reports suggest that the increased proliferation of microglial cells is dependent on ionic membrane currents and in particular on chloride conductances. An unusual chloride ion channel known to be associated with macrophage activation is the chloride intracellular channel-1 (CLIC1). Here we show that Abeta stimulation of neonatal rat microglia specifically leads to the increase in CLIC1 protein and to the functional expression of CLIC1 chloride conductance, both barely detectable on the plasma membrane of quiescent cells. CLIC1 protein expression in microglia increases after 24 hr of incubation with Abeta, simultaneously with the production of reactive nitrogen intermediates and of tumor necrosis factor-alpha (TNF-alpha). We demonstrate that reducing CLIC1 chloride conductance by a specific blocker [IAA-94 (R(+)-[(6,7-dichloro-2-cyclopentyl-2,3-dihydro-2-methyl-1-oxo-1H-inden-5yl)-oxy] acetic acid)] prevents neuronal apoptosis in neurons cocultured with Abeta-treated microglia. Furthermore, we show that small interfering RNAs used to knock down CLIC1 expression prevent TNF-alpha release induced by Abeta stimulation. These results provide a direct link between Abeta-induced microglial activation and CLIC1 functional expression.

An in vitro model for the lepromatous leprosy granuloma: fate of Mycobacterium leprae from target macrophages after interaction with normal and activated effector macrophages.

The lepromatous leprosy granuloma is a dynamic entity requiring a steady influx of macrophages (Mphi) for its maintenance. We have developed an in vitro model to study the fate of Mycobacterium leprae in a LL lesion, with and without immunotherapeutic intervention. Target cells, consisting of granuloma Mphi harvested from the footpads of M. leprae-infected athymic nu/nu mice, were cocultured with normal or IFN-gamma-activated (ACT) effector Mphi. The bacilli were recovered and assessed for viability by radiorespirometry. M. leprae recovered from target Mphi possessed high metabolic activity, indicating a viable state in this uncultivable organism. M. leprae recovered from target Mphi incubated with normal effector Mphi exhibited significantly higher metabolism. In contrast, bacilli recovered from target Mphi cocultured with ACT effector Mphi displayed a markedly decreased metabolic activity. Inhibition by ACT Mphi required an E:T ratio of at least 5:1, a coculture incubation period of 3-5 days, and the production of reactive nitrogen intermediates, but not reactive oxygen intermediates. Neither IFN-gamma nor TNF-alpha were required during the cocultivation period. However, cell-to-cell contact between the target and effector Mphi was necessary for augmentation of M. leprae metabolism by normal effector Mphi as well as for inhibition of M. leprae by ACT effector Mphi. Conventional fluorescence microscopy and confocal fluorescence microscopy revealed that the bacilli from the target Mphi were acquired by the effector Mphi. Thus, the state of Mphi infiltrating the granuloma may markedly affect the viability of M. leprae residing in Mphi in the lepromatous lesion.

Ozone-induced production of nitric oxide and TNF-alpha and tissue injury are dependent on NF-kappaB p50.

Ozone-induced lung injury is associated with increased production of reactive nitrogen intermediates and TNF-alpha, which have been implicated in the pathogenic process. Generation of these mediators is regulated in part by transcription factors, e.g., NF-kappaB and CCAAT/enhancer-binding protein (C/EBP). The present studies used NF-kappaB p50 knockout mice to assess the role of this transcription factor protein in ozone-induced inflammatory mediator production and toxicity. Treatment of wild-type (WT) mice with ozone (0.8 ppm, 3 h) resulted in a rapid increase in NF-kappaB binding activity in alveolar macrophages that peaked after 6-12 h. This response was attenuated in NF-kappaB p50(-)/(-) mice. In WT mice, but not NF-kappaB p50(-)/(-) mice, C/EBP was also markedly increased in macrophages following ozone inhalation. Ozone also induced changes in the mobility of C/EBP in gel shift assays, suggesting alterations in the transcription factor complex that may be important in controlling inflammatory gene expression. Whereas macrophages from WT mice produced increased quantities of nitric oxide and TNF-alpha following ozone inhalation, this was not observed in cells from NF-kappaB p50(-)/(-) mice. Ozone-induced decreases in expression of the anti-inflammatory cytokine IL-10 were also prevented in NF-kappaB p50(-)/(-) mice. In WT mice, ozone inhalation caused an increase in bronchoalveolar lavage protein, a marker of tissue damage. This was not evident in NF-kappaB p50(-)/(-) mice. There was also no evidence of peroxynitrite-mediated lung injury in these mice. These findings demonstrate that NF-kappaB and possibly C/EBP signaling are important in ozone-induced production of reactive nitrogen intermediates and TNF-alpha and in tissue injury.

Impaired production of proinflammatory cytokines and host resistance to acute infection with Trypanosoma cruzi in mice lacking functional myeloid differentiation factor 88.

Studies performed in vitro suggest that activation of Toll-like receptors (TLRs) by parasite-derived molecules may initiate inflammatory responses and host innate defense mechanisms against Trypanosoma cruzi. Here, we evaluated the impact of TLR2 and myeloid differentiation factor 88 (MyD88) deficiencies in host resistance to infection with T. cruzi. Our results show that macrophages derived from TLR2 (-/-) and MyD88(-/-) mice are less responsive to GPI-mucin derived from T. cruzi trypomastigotes and parasites. In contrast, the same cells from TLR2(-/-) still produce TNF-alpha, IL-12, and reactive nitrogen intermediates (RNI) upon exposure to live T. cruzi trypomastigotes. Consistently, we show that TLR2(-/-) mice mount a robust proinflammatory cytokine response as well as RNI production during the acute phase of infection with T. cruzi parasites. Further, deletion of the functional TLR2 gene had no major impact on parasitemia nor on mortality. In contrast, the MyD88(-/-) mice had a diminished cytokine response and RNI production upon acute infection with T. cruzi. More importantly, we show that MyD88(-/-) mice are more susceptible to infection with T. cruzi as indicated by the higher parasitemia and accelerated mortality, as compared with the wild-type mice. Together, our results indicate that T. cruzi parasites elicit an alternative inflammatory pathway independent of TLR2. This pathway is partially dependent on MyD88 and necessary for mounting optimal inflammatory and RNI responses that control T. cruzi replication during the early stages of infection.

Reactive oxygen and nitrogen intermediates contribute to Haemophilus somnus lipooligosaccharide-mediated apoptosis of bovine endothelial cells.

Although Haemophilus somnus causes septicemia and vasculitis in cattle, relatively little is known about how H. somnus affects endothelial cells in vitro. We previously reported that H. somnus lipooligosaccharide (LOS)-induced activation of caspases-3, -8 and -9, and apoptosis of bovine pulmonary artery endothelial cells (BPAEC) in vitro. Previous reports indicate that the generation of reactive oxygen species (ROS) or reactive nitrogen intermediates (RNI) can contribute to the induction of apoptosis. In the present study, we sought to determine whether ROS and RNI are involved in LOS-mediated apoptosis of BPAEC. We found that H. somnus LOS induced the generation of ROS in BPAEC, which was blocked by pretreatment with membrane permeable ROS scavengers, such as dimethylsulfoxide (DMSO) and allopurinol (AP). Addition of DMSO or AP significantly reduced H. somnus LOS-mediated caspase-3 activation. Addition of membrane impermeable ROS scavengers (e.g. catalase and superoxide dismutase), failed to block LOS-mediated caspase-3 activation, suggesting a role for intracellular generation of ROS in LOS-induced apoptosis of BPAEC. Addition of N(G)-nitro-L-arginine methyl ester (L-NAME) or aminoguanidine, which are selective inhibitors of nitric oxide synthase, blocked NO release and significantly reduced caspase-3 activation in LOS treated BPAEC. These data suggest H. somnus LOS triggers endogenous ROS and RNI production by endothelial cells, which contributes to apoptosis.