TNF-alpha In Bronchoalveolar Lavage Fluid Research Articles

Low level laser therapy reduces acute lung inflammation in a model of pulmonary and extrapulmonary LPS-induced ARDS

The present study aimed to investigate the effects low level laser therapy (LLLT) in a LPS-induced pulmonary and extrapulmonary acute respiratory distress syndrome (ARDS) in BALB/c mice. Laser (830nm laser, 9J/cm2, 35mW, 80s per point, 3 points per application) was applied in direct contact with skin, 1h after LPS administration. Mice were distributed in control (n=6; PBS), ARDS IT (n=7; LPS orotracheally 10μg/mouse), ARDS IP (n=7; LPS intra-peritoneally 100μg/mouse), ARDS IT+Laser (n=9; LPS intra-tracheally 10μg/mouse), ARDS IP+Laser (n=9; LPS intra-peritoneally 100μg/mouse). Twenty-four hours after last LPS administration, mice were studied for pulmonary inflammation by total and differential cell count in bronchoalveolar lavage (BAL), cytokines (IL-1beta, IL-6, KC and TNF-alpha) levels in BAL fluid and also by quantitative analysis of neutrophils number in the lung parenchyma. LLLT significantly reduced pulmonary and extrapulmonary inflammation in LPS-induced ARDS, as demonstrated by reduced number of total cells (p<0.001) and neutrophils (p<0.001) in BAL, reduced levels of IL-1beta, IL-6, KC and TNF-alpha in BAL fluid and in serum (p<0.001), as well as the number of neutrophils in lung parenchyma (p<0.001). LLLT is effective to reduce pulmonary inflammation in both pulmonary and extrapulmonary model of LPS-induced ARDS.

A new RAGE blocker, low anti‐coagulant 2‐O,3‐O desulfated heparin (ODSH), diminishes smoke‐induced pulmonary inflammation

Research performed in our lab has implicated the receptor for advanced glycation end-products (RAGE), a pattern recognition receptor, in pulmonary inflammation induced by tobacco smoke, diesel particulate matter, and hyperoxia. Recent work has also demonstrated that low anti-coagulant 2-O,3-O-desulfated heparin (ODSH) may function as an effective blocker of RAGE-ligand interactions as evidenced by inhibition of RAGE ligation with its disparate ligands including CML-BSA (AGE), HMGB-1, and S100b. The current study sought to characterize the anti-inflammatory effects of ODSH in BALB/c mice exposed to acute tobacco smoke. Mice were anesthetized and administered PBS or cigarette smoke extract (CSE, 0.02 cigarettes per dose) +/− 50 ug ODSH via nasal installation. Bronchoalveolar lavage fluid (BALF) was obtained 48 hours after exposure and analyzed. CSE induced a significant increase in total protein, total leukocyte count, PMN count, and cytokine concentration including TNF-alpha in BALF. ODSH administration resulted in a significant decrease in CSE-induced total protein, PMN quantity, and cytokine concentration in BALF compared to controls. The data show that ODSH blockade of RAGE and parallel signaling mechanisms can diminish smoke-induced pro-inflammatory events. Work was supported by the Flight Attendant¡¦s Medical Research Institute (FAMRI, PRR) and a BYU Mentoring Environment Grant (PRR).

Mechanical ventilation with high tidal volume induces inflammation in patients without lung disease

IntroductionMechanical ventilation (MV) with high tidal volumes may induce or aggravate lung injury in critical ill patients. We compared the effects of a protective versus a conventional ventilatory strategy, on systemic and lung production of tumor necrosis factor-α (TNF-α) and interleukin-8 (IL-8) in patients without lung disease.MethodsPatients without lung disease and submitted to mechanical ventilation admitted to one trauma and one general adult intensive care unit of two different university hospitals were enrolled in a prospective randomized-control study. Patients were randomized to receive MV either with tidal volume (VT) of 10 to 12 ml/kg predicted body weight (high VT group) (n = 10) or with VT of 5 to 7 ml/kg predicted body weight (low VT group) (n = 10) with an oxygen inspiratory fraction (FIO2) enough to keep arterial oxygen saturation >90% with positive end-expiratory pressure (PEEP) of 5 cmH2O during 12 hours after admission to the study. TNF-α and IL-8 concentrations were measured in the serum and in the bronchoalveolar lavage fluid (BALF) at admission and after 12 hours of study observation time.ResultsTwenty patients were enrolled and analyzed. At admission or after 12 hours there were no differences in serum TNF-α and IL-8 between the two groups. While initial analysis did not reveal significant differences, standardization against urea of logarithmic transformed data revealed that TNF-α and IL-8 levels in bronchoalveolar lavage (BAL) fluid were stable in the low VT group but increased in the high VT group (P = 0.04 and P = 0.03). After 12 hours, BALF TNF-α (P = 0.03) and BALF IL-8 concentrations (P = 0.03) were higher in the high VT group than in the low VT group.ConclusionsThe use of lower tidal volumes may limit pulmonary inflammation in mechanically ventilated patients even without lung injury.Trial RegistrationClinical Trial registration: NCT00935896

Association of Tumor Necrosis Factor-.ALPHA. with Fever and Pulmonary Lesion Score in Pigs Experimentally Infected with Swine Influenza Virus Subtype H1N2

The relationship between tumor necrosis factor (TNF)-alpha level, body temperature, and pulmonary lesion score was determined in 3-week-old pigs inoculated intranasally with swine influenza virus (SIV) subtype H1N2. The expression of TNF-alpha was measured in bronchoalveolar lavage (BAL) fluids by enzyme-linked immunosorbent assay and lung tissues by immunohistochemistry. In BAL fluid, TNF-alpha concentration was maximal at 1 days post-inoculation (dpi), declined markedly by 3 dpi (P<0.05) and steady thereafter. Mean rectal temperatures were above 40 degrees C for the infected groups at 1 dpi and declined markedly by 3 dpi. The body temperatures were correlated with the levels of TNF-alpha in BAL fluids from pigs experimentally infected with SIV (r(s)=0.9754, P<0.05). The pulmonary lesion scores were correlated with the means of positive cells by immunohistochemistry for TNF- alpha (r(s)=0.9949, P<0.001). The results suggest that the expression of TNF-alpha protein plays an important role in induction of pulmonary lesion and clinical sign such as fever in SIV infection.

Expression levels of immune markers in Actinobacillus pleuropneumoniae infected pigs and their relation to breed and clinical symptoms

BackgroundIn pigs little is known about the role of innate immune defence in bacterial infections of the respiratory tract, despite their major role in pig production. In the present study we characterized and compared in vitro and in vivo activation of immune markers of different pig breeds 7 days before, and 4 and 21 days after an experimental aerosol infection with Actinobacillus (A.) pleuropneumoniae.ResultsIn vitro stimulation of bronchoalveolar lavage fluid (BALF) and blood leukocytes with A. pleuropneumoniae, Streptococcus suis, PMA and LPS led to production of different amounts of H2O2, NO and TNF-α, depending on the stimulus, individual, breed and time of infection. Generally, significant responses to in vitro stimulation were observed only in blood leukocytes, whereas the alveolar macrophages showed a high basal activation. In addition, the production of haptoglobin and cytokines (TNF-α, IFN-γ and IL-10) in vivo was measured in plasma and BALF. Plasma haptoglobin levels mirrored the clinical manifestations at 4 days post-infection. In plasma and BALF TNF-α could not be detected, whereas variable levels of IFN-γ were found at pre- and post-infection times. IL-10 was found in some plasma but in none of the BALF samples. The different expression levels in individuals within the breeds correlated for some markers with the severity of clinical manifestations, e.g. H2O2, plasma haptoglobin and BALF IFN-γ for German Landrace pigs.ConclusionOur findings revealed differences in the activation of the immune markers with respect to infection time, individuals and breeds. Moreover, results showed different correlation grades between the immune markers produced in vitro or in vivo and the clinical manifestations. Further analyses will have to show whether these markers may serve as correlates of protection against porcine respiratory infections.

Inhibition of tumor necrosis factor-α reduces alveolar septal cell apoptosis in passive smoking rats

Recent studies have revealed that lung cell apoptosis plays an important role in pathogenesis of cigarette-induced chronic obstructive pulmonary disease (COPD). Tumor necrosis factor alpha (TNF-alpha) is one of the most important cytokines which are involved in COPD. This study aimed at investigating the influence of its inhibitor, recombinant human necrosis factor-alpha receptor II:IgG Fc fusion protein (rhTNFR:Fc) on alveolar septal cell apoptosis in passive smoking rats. Forty-eight rats were randomly divided into a normal control group, a passive smoking group, an rhTNFR:Fc intervention group and a sham intervention group. The passive smoking rats were treated by exposure to cigarette smoking daily for 80 days. After smoking for one month the rhTNFR:Fc intervention group was treated with rhTNFR:Fc by subcutaneous injection, the sham intervention group injected subcutaneously with a neutral preparation (normal saline 0.1 ml, manicol 0.8 ml, cane sugar 0.2 mg, Tris 0.024 mg as a control. Lung function was determined and the levels of TNF-alpha in serum and broncho-alveolar lavage fluid (BALF) were measured with enzyme-linked immunosorbnent assay (ELISA). Lung tissue sections stained by hematoxylin and eosin (HE) were observed for study of morphological alternations. Mean linear intercept (MLI) and mean alveolar numbers (MAN) were measured and the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method was carried out to determine the percentage of positive cells and distribution of apoptotic cells. Increased MLI and decreased MAN were found in the passive smoking group compared with both the normal control group and the rhTNFR:Fc intervention group (P < 0.05). Forced expiratory volume in 0.3 second (FEV 0.3)/forced vital capacity (FVC) and peak expiratory flow (PEF) were lower in the passive smoking group than that in the normal control group (P < 0.05). Compared with the sham intervention group, FEV 0.3/FVC and PEF increased in the rhTNFR:Fc intervention group (P < 0.05). The levels of TNF-alpha in serum were higher in the passive smoking group than that in the normal control group (P < 0.05) and rhTNFR:Fc intervention group (P < 0.05). Significant differences were found between the levels of TNF-alpha in the serum of the rhTNFR:Fc intervention group and sham intervention group (P < 0.05). The levels of TNF-alpha in BALF were higher in the passive smoking group than that in the normal control group (P < 0.05), but no significant differences of TNF-alpha levels in BALF were found between the passive smoking group and rhTNFR:Fc intervention group. The number of TUNEL positive cells in alveolar septa was significantly increased in the passive smoking group as compared with the normal control group and the rhTNFR:Fc intervention group (P < 0.05). This study provides preliminary evidence that rhTNFR:Fc may interfere with TNF-alpha and reduce alveolar septal apoptosis in smoking rats.

The fall in exhaled nitric oxide with ventilation at low lung volumes in rabbits: An index of small airway injury

The mechanisms involved in the fall of exhaled nitric oxide (NOe) concentration occurring in normal, anesthetized open chest rabbits with prolonged mechanical ventilation (MV) at low lung volume have been investigated. NOe, pH of exhaled vapor condensate, serum prostaglandin E(2), and F(2alpha), tumor necrosis factor (TNF-alpha), PaO(2), PaCO(2), pHa, and lung mechanics were assessed before, during, and after 3-4h of MV at zero end-expiratory pressure (ZEEP), with fixed tidal volume (9 ml kg(-1)) and frequency, as well as before and after 3-4h of MV on PEEP only. Lung histology and wet-to-dry ratio (W/D), and prostaglandin and TNF-alpha in bronchoalveolar lavage fluid (BALF) were also assessed. While MV on PEEP had no effect on the parameters above, MV on ZEEP caused a marked fall (45%) of NOe, with a persistent increase of airway resistance (45%) and lung elastance (12%). Changes in NOe were independent of prostaglandin and TNF-alpha levels, systemic hypoxia, hypercapnia and acidosis, bronchiolar and alveolar interstitial edema, and pH of exhaled vapor condensate. In contrast, there was a significant relationship between the decrease in NOe and bronchiolar epithelial injury score. This indicates that the fall in NOe, which occurs in the absence of an inflammatory response, is due to the epithelial damage caused by the abnormal stresses related to cyclic opening and closing of small airways with MV on ZEEP, and suggests its use as a sign of peripheral airway injury.

Dynamic expression of tumor necrosis factor-α and vascular endothelial growth factor in rat model of pulmonary emphysema induced by smoke exposure

In order to explore the roles of tumor necrosis factor-alpha (TNF-alpha) and vascular endothelial growth factor (VEGF) in the pathogenesis of pulmonary emphysema, male Wistar rats were randomized into group A(1), group A(2.5) and group A(4), each with smoke exposure for 1 month, 2.5 months or 4 months, respectively. Group B(1), group B(2.5) and group B(4) were used as non smoking controls at corresponding time points. TNF-alpha in bronchoalveolar lavage fluid (BALF) and expression of VEGF in lung tissue was determined by ELISA or by SABC immunohistochemistry assay either. Lung slices were stained with hematoxylin and eosin (HE). Results showed that in animal with smoke exposure the mean linear interceptor (Lm), an index of pulmonary emphysema and the content of TNF-alpha in BALF increased gradually, on contrary, the expression of VEGF in lung tissue decreased (P<0.05). This phenomenon was not obvious in animals without smoke exposure. Lm was negatively correlated to the VEGF expression (gamma=-0.81, P<0.01) and positively correlated to TNF-alpha concentration (gamma = 0.52, P<0.004), which implies that smoke exposure decreased the expression of VEGF and increased the expression of TNF-alpha. It is plausible to speculate that the imbalance of TNF-alpha and VEGF may play an important role in the pathogenesis of smoke-induced pulmonary emphysema.

Reduced Th1 response in the lungs of HLA-DRB1*0301 patients with pulmonary sarcoidosis

To investigate why human leukocyte-associated antigen-DRB1*0301 (HLA-DRB1*0301) positive Scandinavian patients have a better prognosis than HLA-DRB1*0301 negative patients, the present authors examined patterns of cytokine expression in bronchoalveolar lavage (BAL) cells and BAL fluid (BALF) from patients with pulmonary sarcoidosis and controls. Using real-time PCR, the mRNA expression of selected cytokines in BAL cells from newly diagnosed, untreated nonsmoking patients (n=25) and controls (n=11) was quantified. Cytokine protein levels in BALF from patients (n=34) and controls (n=11) were assessed using cytometric bead array. The patients were evaluated and stratified into two subgroups: HLA-DRB1*0301 positive (all with an acute onset) and HLA-DRB1*0301 negative (all with an insidious onset). When comparing patients and controls, BAL cells of the patients expressed significantly higher levels of interferon (IFN)-gamma and interleukin (IL)-10 mRNA. There were significantly decreased IFN-gamma and tumour necrosis factor (TNF)-alpha mRNA levels, and a tendency toward higher levels of transforming growth factor-beta1 mRNA in HLA-DRB1*0301 positive compared with HLA-DRB1*0301 negative patients. Protein levels of IL-1beta, IL-2, IL-6, IL-12p70 and TNF-alpha in BALF were significantly higher in patients. HLA-DRB1*0301 positive patients exhibited tendencies to lower levels of most cytokines in BALF. In conclusion, the present data show a reduced expression of T-helper cell type-1 cytokines in human leukocyte-associated antigen-DRB1*0301 positive patients, which may relate to their good prognosis.

Endotoxin tlerance inhibits lipopolysaccharide-initiated acute pulmonary inflammation and lung injury in rats by the mechanism of nuclear factor-kappaB.

In this study, the effect of endotoxin tolerance on lipopolysaccharide (LPS)-initiated pulmonary inflammation, the local production of tumour necrosis factor-alpha (TNF-alpha) and the cytokine-induced neutrophil attractant (CINC), as well as the activation of nuclear factor-kappaB (NF-kappaB) and its subunit composition, were examined in vivo. Endotoxin tolerance was reproduced by four consecutive daily intraperitoneal injections of 0.6 mg/kg of Escherichia coli 055:B5 LPS. Compared with control rats, endotoxin-tolerant rats failed to increase the permeability of pulmonary microvascular or recruit neutrophil to lung tissue upon restimulation with 6 mg/kg of LPSs. Pretreatment with LPSs inhibited the protein level of TNF-alpha in bronchoalveolar lavage fluid (BALF) and mRNA expression of CINC in lung tissue in response to subsequent LPS stimulation. These changes were accompanied by the suppression of activation of NF-kappaB, including the low level of total amount of DNA-binding activity and high percentage of non-transactive p50 homodimers. These data demonstrate that endotoxin tolerance can alleviate the LPS-induced acute neutrophilic pulmonary inflammation in rats and can inhibit the proinflammatory cytokines in lung and suggest that endotoxin tolerance might result from the unresponsiveness of NF-kappaB and persistent high percentage of p50 homodimers. Therefore, the phenomenon of endotoxin tolerance might be used as a strategy for the prevention or treatment of LPS-associated acute respiratory distress syndrome in which excessive or dysregulated inflammation leads to acute lung injury.

Temporal association between airway hyperresponsiveness and airway eosinophilia in ovalbumin-sensitized mice.

The temporal association between airway inflammation and airway hyperresponsiveness (AHR) has been analyzed in BALB/c mice sensitized to, and subsequently exposed to, a single intranasal challenge of ovalbumin (OVA). In OVA-sensitized/challenged animals only a small increase in responsiveness to methacholine (MCh) was seen at 8 h, peaked at 24 to 48 h, and resolved by 96 h. An early bronchoalveolar lavage fluid (BALF) neutrophil infiltrate (peaking at 8 h postchallenge; approximately 72% total cells was observed) that returned to baseline by 48 h. BALF eosinophil numbers did not increase until 48 h (approximately 32% of total cells), peaked at 96 h (approximately 38% total cells), and remained elevated at 8 d (approximately 27% total cells). Airway tissue eosinophilia preceded changes in BALF. Eosinophil peroxidase (EPO) levels in BALF were elevated in OVA-sensitized/challenged mice at 48 h only. BALF TNF-alpha levels peaked at 8 h, whereas IL-5 and IL-4 levels peaked at 24 h. IL-13 levels were increased at both 24 and 48 h. Mucus-positive cells were not observed in the airway epithelium until 48 h. Administration of IL-5 or VLA-4 antibody prior to OVA challenge prevented the development of AHR in sensitized mice as well as BALF and tissue eosinophilia. These data identify a temporal association between Th2 cytokine production, tissue eosinophil infiltration and activation, and, importantly, both the development and resolution kinetics of AHR. Moreover, the antibody studies further support the association of eosinophilia with the pathogenesis of AHR.

Different ventilation strategies affect lung function but do not increase tumor necrosis factor-alpha and prostacyclin production in lavaged rat lungs in vivo.

Using an in vivo animal model of surfactant deficiency, the authors compared the effect of different ventilation strategies on oxygenation and inflammatory mediator release from the lung parenchyma. In adult rats that were mechanically ventilated with 100% oxygen, acute lung injury was induced by repeated lung lavage to obtain an arterial oxygen partial pressure < 85 mmHg (peak pressure/positive end-expiratory pressure [PEEP] = 26/6 cm H2O). Animals were then randomly assigned to receive either exogenous surfactant therapy, partial liquid ventilation, ventilation with high PEEP (16 cm H2O), ventilation with low PEEP (8 cm H2O), or ventilation with an increase in peak inspiratory pressure (to 32 cm H2O; PEEP = 6 cm H2O). Two groups of healthy nonlavaged rats were ventilated at a peak pressure/PEEP of 32/6 and 32/0 cm H2O, respectively. Blood gases were measured. Prostacyclin (PGI2) and tumor necrosis factor-alpha (TNF-alpha) concentrations in serum and bronchoalveolar lavage fluid (BALF) as well as protein concentration in BALF were determined after 90 and 240 min and compared with mechanically ventilated and spontaneously breathing controls. Surfactant, partial liquid ventilation, and high PEEP improved oxygenation and reduced BALF protein levels. Ventilation with high PEEP at high mean airway pressure levels increased BALF PGI2 levels, whereas there was no difference in BALF TNF-alpha levels between groups. Serum PGI2 and TNF-alpha levels did not increase as a result of mechanical ventilation when compared with those of spontaneously breathing controls. Although alveolar protein concentration and oxygenation markedly differed with different ventilation strategies in this model of acute lung injury, there were no indications of ventilation-induced systemic PGI2 and TNF-alpha release, nor of pulmonary TNF-alpha release. Mechanical ventilation at high mean airway pressure levels increased PGI2 levels in the bronchoalveolar lavage-accessible space.

Airway exposure to bacterial superantigen (SEB) induces lymphocyte-dependent airway inflammation associated with increased airway responsiveness--a model for non-allergic asthma.

Although immunological consequences of systemic superantigen administration have been extensively studied, the effects of local mucosal exposure to superantigens are not well defined. The purpose of this study was to delineate the type of immune response triggered by superantigen exposure to the airway mucosa in mice. In dose-response experiments we determined a low dose of staphylococcal enterotoxin B (SEB) that triggered an inflammatory response characterized by mucosal and airway recruitment of lymphocytes, eosinophils and neutrophils together with elevated levels of IL-4, but not IFN-gamma, in bronchoalveolar lavage (BAL) fluids. TCR Vbeta analysis revealed that superantigen-responsive and -non-responsive T cells were equally recruited into the airways. SEB markedly enhanced the frequency of TNF-alpha-positive BAL macrophages as well as the amount of TNF-alpha in BAL fluids. These responses were associated with the development of increased airway responsiveness (AR) in SEB-treated mice. This effect occurred in an antibody-independent fashion. Furthermore, this type of response was observed in IgE-high responder BALB/c as well as in IgE-low/intermediate responder C57BL/6 mice. The development of increased AR was CD4+ T cell dependent as shown by transfer experiments into BALB/c nu/nu mice. These results suggest that the local immune response following mucosal superantigen administration triggers a unique inflammatory response in the airways resembling many features of "intrinsic asthma".

Ethanol Inhibits the Potentiation of Endotoxin by Dibutyryl cAMP and 2‐Methylthio ATP In Vivo

Ethanol (ETOH) selectively suppressed Escherichia coli endotoxin lipopolysaccharide (LPS)-stimulated, but not dibutyryl cAMP (db-cAMP)-stimulated upregulation of inducible nitric oxide synthase (iNOS) in rat alveolar macrophages (AMs) in vivo (Zhao et al., Alcohol. Clin. Exp. Res. 21:1062-1074, 1997). LPS-induced stimulation of iNOS is inhibited in vitro by db-cAMP and purine-2-Y (P2Y) receptor-mediated agonists. We examined the effect of ETOH on this interaction in rat lung AMs in vivo. Two hours after co-administration of LPS [0.6 mg/kg, intratracheal (i.t.)] with db-cAMP (0.1 mg/kg, i.t.) or 2-methylthio-adenosine-triphosphate (2-mes-ATP) to Sprague-Dawley rats (225 to 250 g) (n = 10 to 24/gp) iNOS messenger ribonucleic acid (mRNA), iNOS protein, and nitrate and nitrite anions [reactive nitrogen intermediates (RNIs)] in bronchoalveolar lavage fluid (BALf), and the ex vivo incubates of AMs were increased more than when these compounds were given individually to the rats. Co-administration of LPS with the autacoids did not affect LPS-stimulated increases of tumor necrosis factor-alpha (TNF alpha) mRNA, but inhibited LPS-stimulated BALf TNF alpha protein and attenuated LPS-mediated decreases of cell-associated TNF alpha. Pretreatment of rats with ETOH (4.5 g/kg, i.p.) or diethyidithiocarbamate (DETC; 5 mg/kg, i.t.), an inhibitor of the nuclear transcription factor NF kappa B (NF-kappaB), 30 min before co-administration of LPS with the autacoids restored iNOS mRNA levels to that obtained with the autacoid alone. Pretreatment of rats with ETOH decreased iNOS protein and RNI below that produced by either compound given individually to the rats. Although pretreatment of rats with DETC also decreased iNOS protein and RNI levels produced by LPS, the levels of both substances were elevated above that of the autacoid alone, LPS-induced upregulation of iNOS mRNA was associated with elevated levels of p65/p50 NF-kappaB in the nucleus of AMs. Neither db-cAMP or 2-mes-ATP affected LPS-stimulated NF-kappaB-DNA binding. Moreover, ETOH and DETC inhibited LPS-stimulated NF-kappaB-DNA binding. We conclude that in rat AMs in vivo: (1) both db-cAMP and P2Y-receptor stimulation summate with, rather than inhibit, LPS-induced upregulation of the iNOS mRNA, protein, and RNI; (2) ETOH and DETC inhibit LPS-induced upregulation of iNOS mRNA only when stimulated through the NF-kappaB pathway; and (3) ETOH inhibits both autacoid and LPS-stimulated formation of iNOS protein by a mechanism independent of its ability to suppress iNOS transcription.

Ethanol Inhibits Inducible Nitric Oxide Synthese Transcription and Post‐Transcriptional Processes in Vivo

The effects of ethanol (ETOH) on post-transcriptional regulation of inducible nitric oxide synthase (iNOS) in vivo has not been demonstrated. We examined the effect of ETOH on iNOS mRNA, protein, and the production of the nitrate and nitrite anion (RNI) in rat lung alveolar macrophages (AM) in vivo when stimulated by lipopolysaccharide (LPS) and dibutyryl cyclic AMP (DB-cAMP). Sprague-Dawley rats (225-250 g) (n = 5-7/gp) were given intratracheal LPS (0.6 mg/kg) or DB-cAMP (0.1 and 1 mg/kg) 30 min after ETOH (4 mg/kg, intraperitoneally (ip)) or phosphate-buffered sterile saline (PBS) (5 ml/kg, ip) or pyrrolidine dithiocarbamate (PDTC 10 mg/kg, intratracheally) or 15 min after diethyl dithiocarbamate (DETC) (5 mg/kg, intratracheally). At selected times after administration of LPS or DB-cAMP, the animals were anesthetized, the lungs with the heart attached were removed and the lungs subjected to bronchoalveolar lavage (BAL). The BAL fluid was assayed for tumor necrosis factor alpha (TNF alpha) and RNI. The BAL fluid AM were isolated and analyzed for iNOS mRNA and protein with competitor-equalized polymerase chain reaction (PCR) and Western blots, respectively. The ex vivo incubates of AM were assayed for RNI and TNF alpha. LPS and DB-cAMP each increased iNOS mRNA and protein in AM and RNI in the BAL fluid and ex vivo incubates of AM. However, the peak of the increase of iNOS mRNA occurred at 2 hr for DB-cAMP and 4 to 6 hr for LPS. Only LPS increased the concentrations of TNF alpha in BAL fluid and ex vivo incubates of AM. ETOH attenuated LPS-mediated up-regulation of iNOS mRNA and TNF alpha and iNOS protein and RNI produced by the AM. In contrast, pretreatment of rats with ETOH did not affect DB-cAMP-mediated increases of iNOS mRNA in AM at any time but suppressed the amount of iNOS protein and RNI produced in DB-cAMP-stimulated AM. DETC, but not PDTC, attenuated LPS-mediated up-regulation of iNOS mRNA without effects on that produced by DB-cAMP. Since ETOH and DETC, but not PDTC, suppressed LPS-mediated but not DB-cAMP-mediated transcription of iNOS, we conclude that two distinct pathways exist for induction of iNOS mRNA by these agonists. ETOH and DETC may inhibit the LPS-mediated activation pathway by acting as antioxidants. Also, since ETOH inhibited DB-cAMP-mediated increases in iNOS protein without affecting iNOS mRNA ETOH also acts at a post-transcriptional or translational site to inhibit iNOS protein in rat AM in vivo.

Effects of granulocyte and granulocyte-macrophage colony-stimulating factors in a neutropenic murine model of trichosporonosis.

We produced disseminated trichosporonosis in a neutropenic murine model with Trichosporon asahii, which was identified by DNA relatedness analysis. We then assessed the efficacy of granulocyte colony-stimulating factor (G-CSF) (30 to 100 microg/kg of body weight per day) and granulocyte-macrophage colony-stimulating factor (GM-CSF) (0.8 to 2 microg/kg x day). The administration of G-CSF either before or after infection improved the survival rate from less than 25% up to 100% (P < 0.05). The effects of G-CSF on organ clearance and histological examinations were most remarkable in the lungs. The levels of tumor necrosis factor alpha (TNF-alpha) in bronchoalveolar lavage fluid (BALF) of neutropenic and G-CSF-pretreated mice were 60 +/- 6 ng/ml and 18 +/- 6 pg/ml, respectively, at 24 h after infection. Immunohistologically, alveolar macrophages proved to be the main source of TNF-alpha in BALF. GM-CSF increased neutrophil counts less significantly than did G-CSF and increased the lethality (P < 0.05) with a high level of TNF-alpha in BALF. Expecting to inhibit TNF-alpha, we administered anti-TNF-alpha intraperitoneally at the dose completely inhibiting TNF-alpha in plasma (2 x 10(4) U), but the TNF-alpha level in BALF and the lethality increased. Though the number of neutrophils at the early stage of infection appeared to be the most critical, the results suggest that other host defense mechanisms, such as TNF-alpha overproduction in the lungs, have an important role in the prognosis of trichosporonosis.

Early-type hypersensitivity-associated airway inflammation and eosinophilia induced by Dermatophagoides farinae in sensitized mice.

In a murine ear-swelling model, we demonstrated a unique hypersensitivity response and defined it as early-type hypersensitivity (ETH). ETH was characterized by increased vasopermeability and edematous change that occurred within 1 h at the site of Ag challenge. In this study, intranasal challenge with Dermatophagoides farinae (Df) on Df-sensitized BALB/c mice induced an ETH response in the lungs. The lung ETH was manifested by an increase in wet lung weight, production of TNF-alpha in bronchoalveolar lavage fluids, and hyperemia and edematous change around vessels of small airways 1 h after Ag provocation. The challenged animals subsequently developed airway inflammation, beginning with a neutrophilic infiltrate which was followed by lymphocytes and eosinophils. The Df-induced eosinophilia was Ag-specific and maximal at 48 h after challenge. At this time, the trachea from sensitized mice also exhibited hyperreactivity to carbachol. Pretreatment with anti-CD4+ mAb significantly decreased the recruitment of eosinophils in bronchoalveolar lavage fluids. An enhanced expression of pulmonary endothelial vascular cell adhesion molecule-1 was noted as early as 6 h after challenge. Anti-Df Abs of IgG class, but not IgE class, were detected in Df-immunized mice at the time of challenge. Furthermore, Df challenge induced a stronger eosinophil response in BALB/c mice (H-2d) than in B10.BR (H-2k) mice. B10.BR mice also did not exhibit pulmonary edema or ETH of ear swelling 1 h after challenge. These data suggest that an ETH-associated 1 h pulmonary edematous change was induced by intranasal challenge of Df in Df-sensitized mice, and that the ETH might contribute to the development of subsequent pulmonary inflammation and eosinophilia.

Effect of Arsenic Exposure on Alveolar Macrophage Function .2. Effect of Slightly Soluble Forms of as(III) and as(V)

The pulmonary toxicity of a substance depends on a number of chemical and physical characteristics, including the solubility of the compounds. In the lung, insoluble forms of metals may be more tumorigenic than soluble forms despite the fact that this effect has not been quantitated and the mechanism of action has not been elucidated. The toxic effects of slightly soluble forms of As(III) and As(V) were evaluated by determining alteration in function of pulmonary alveolar macrophages (PAM) following in vivo and in vitro exposure. Male Sprague-Dawley rats were used throughout. Twenty-four hours following intratracheal instillation of 1 mg/kg (as arsenic) of either arsenic trisulfide (As(III)) or calcium arsenate (As(V)), PAM were lavaged and analyzed for alterations in superoxide (O-2(-)), and tumor necrosis factor (TNF-alpha) production. There were no differences in bronchoalveolar lavage fluid TNF-alpha. PAM. lavaged from As(V)-exposed animals showed significant increases in O-2(-) production and in basal release of TNF-alpha. PAM lavaged from animals receiving As(III) did not show significant alterations. To test the direct effects of arsenic, PAM were lavaged from control animals and exposed to concentrations of 0,1 to 300 mu g/ml arsenic in vitro for up to 24 hr. Doses used were not cytotoxic to PAM, since LDH release was not significantly increased. Significant dose-dependent inhibition of O-2(-) production was only evident after 24 hr exposure to arsenicals. Both As(III) and As(V) produced inhibition at concentrations of 10 mu g/ml. Suppression of LPS-induced release of TNF-alpha also occurred at similar concentrations for both arsenicals (4-5 mu g/ml). Neither arsenical inhibited prostaglandin E(2) production. Measurement of soluble arsenic concentrations indicated dissolution of the compounds could not account for all of the effects seen. Arsenic-induced alteration in PAM function may compromise host defense.

Endotoxin responsiveness and grain dust-induced inflammation in the lower respiratory tract

To identify the role of endotoxin responsiveness in grain dust-induced airway disease, we used two models of extotoxin hyporesponsiveness to perform inhalation exposure studies in mice. In the first model, we investigated whether genetic resistance to endotoxin would alter the inflammatory response to inhaled grain dust by comparing the inflammatory response in the lower respiratory tract of endotoxin-sensitive and -resistant male mice after inhalation of pyrogen-free saline, corn dust extract (CDE), sterile CDE (SCDE), or lipopolysaccharide (LPS). Endotoxin-sensitive and -resistant mice were exposed for 4 h to nebulized solutions of LPS, SCDE, or CDE. Another group of endotoxin-sensitive and -resistant mice was sham exposed for 4 h to nebulized sterile saline. Dose-response relationships for endotoxin were explored for LPS, SCDE, and CDE. Bronchoalveolar lavage (BAL) 5 h after the start of exposure demonstrated a higher concentration of total cells, neutrophils (PMNs), and tumor necrosis factor-alpha (TNF-alpha) in BAL fluid after inhalation of CDE, SCDE, or LPS in endotoxin-sensitive than in endotoxin-resistant mice. Whereas endotoxin-sensitive mice demonstrated a dose-response relationship between the endotoxin concentration in each of the solutions and the concentration of cells, PMNs, and TNF-alpha in BAL fluid, concentrations of TNF-alpha were significantly higher only in BAL fluid of endotoxin-resistant mice exposed to higher concentrations of SCDE or CDE. In the second model, we investigated whether acquired endotoxin tolerance would alter the inflammatory response to SCDE.(ABSTRACT TRUNCATED AT 250 WORDS)