Role of IL-18 in acute lung inflammation.

We have examined the role of IL-18 after acute lung inflammation in rats caused by intrapulmonary deposition of IgG immune complexes. Constitutive IL-18 mRNA and protein expression (precursor form, 26 kDa) were found in normal rat lung, whereas in inflamed lungs, IL-18 mRNA was up-regulated; in bronchoalveolar (BAL) fluids, the 26-kDa protein form of IL-18 was increased at 2-4 h in inflamed lungs and remained elevated at 24 h, and the "mature" protein form of IL-18 (18 kDa) appeared in BAL fluids 1-8 h after onset of inflammation. ELISA studies confirmed induction of IL-18 in inflamed lungs (in lung homogenates and in BAL fluids). Prominent immunostaining for IL-18 was found in alveolar macrophages from inflamed lungs. When rat lung macrophages, fibroblasts, type II cells, and endothelial cells were cultured in vitro with LPS, only the first two produced IL-18. Intratracheal administration of rat recombinant IL-18 in the lung model caused significant increases in lung vascular permeability and in BAL content of neutrophils and in BAL content of TNF-alpha, IL-1beta, and cytokine-induced neutrophil chemoattractant, whereas intratracheal instillation of anti-IL-18 greatly reduced these changes and prevented increases in BAL content of IFN-gamma. Intratracheal administration of the natural antagonist of IL-18, IL-18 binding protein, resulted in suppressed lung vascular permeability and decreased BAL content of neutrophils, cytokines, and chemokines. These findings suggest that endogenous IL-18 functions as a proinflammatory cytokine in this model of acute lung inflammation, serving as an autocrine activator to bring about expression of other inflammatory mediators.     

Differential effects of general anesthesia on cGMP-mediated pulmonary vasodilation.

We investigated the effects of an intravenous (pentobarbital sodium) and an inhalational (halothane) general anesthetic on guanosine 3',5'-cyclic monophosphate- (cGMP) mediated pulmonary vasodilation compared with responses measured in the conscious state. Multipoint pulmonary vascular pressure-flow plots were generated in the same nine dogs in the fully conscious state, during pentobarbital sodium anesthesia (30 mg/kg iv), and during halothane anesthesia (approximately 1.2% end tidal). Continuous intravenous infusions of bradykinin (2 micrograms.kg-1.min-1) and sodium nitroprusside (5 micrograms.kg-1.min-1) were utilized to stimulate endothelium-dependent and -independent cGMP-mediated pulmonary vasodilation, respectively. In the conscious state, both bradykinin and nitroprusside decreased (P less than 0.01) the pulmonary vascular pressure gradient (pulmonary arterial pressure-pulmonary arterial wedge pressure) over the entire range of flows studied; i.e., bradykinin and nitroprusside caused active flow-independent pulmonary vasodilation. Pulmonary vasodilator responses to bradykinin (P less than 0.01) and nitroprusside (P less than 0.05) were also observed during pentobarbital anesthesia. In contrast, during halothane anesthesia, the pulmonary vasodilator responses to both bradykinin and nitroprusside were abolished. These results indicate that, compared with the conscious state, cGMP-mediated pulmonary vasodilation is preserved during pentobarbital anesthesia but is abolished during halothane anesthesia.     

FDG-PET imaging of pulmonary inflammation in healthy volunteers after airway instillation of endotoxin.

Recent studies indicate that a focal, limited, inflammatory response can be safely elicited after direct bronchial instillation of small doses of endotoxin into a single lung segment. Because the radiotracer [18F]fluorodeoxyglucose ([18F]FDG) is taken up at accelerated rates within inflamed tissues, we hypothesized that we could detect and quantify this regional inflammatory response with positron emission tomography (PET). We imaged 18 normal volunteers in a dose-escalation study with 3 endotoxin dosing groups (n = 6 in each group): 1 ng/kg, 2 ng/kg, and 4 ng/kg. Endotoxin was instilled by bronchoscopy into a segment of the right middle lobe, with imaging performed approximately 24 h later, followed by bronchoalveolar lavage (BAL). A "subtraction imaging analysis" was performed in the highest dose cohort to identify the area of inflammation, using the preendotoxin scan as a baseline. BAL neutrophil counts were significantly higher in the highest dose group compared with the other two groups (1,413 +/- 625 vs. 511 +/- 396 and 395 +/- 400 cells/mm3; P < 0.05). Autoradiography performed on cells harvested by BAL showed specific [3H]deoxyglucose ([3H]DG) uptake limited to neutrophils. In vitro [3H]DG uptake in BAL neutrophils in the 4 ng/kg dose group (but not in the 2 ng/kg group) was statistically greater than in peripheral blood neutrophils obtained before endotoxin instillation. The rate of [18F]FDG uptake was greatest in the 4 ng/kg group, with a consistent, statistically significant increase in the rate of uptake after endotoxin instillation compared with baseline. We conclude that the inflammatory response to low-dose endotoxin in a single lung segment can be visualized and quantified by imaging with FDG-PET.     

Essential contribution of monocyte chemoattractant protein-1/C-C chemokine ligand-2 to resolution and repair processes in acute bacterial pneumonia

Neutrophil infiltration is the first step in eradication of bacterial infection, but neutrophils rapidly die after killing bacteria. Subsequent accumulation of macrophage lineage cells, such as alveolar macrophages (AMs), is essential to remove dying neutrophils, which are a source of injurious substances. Macrophage lineage cells can promote tissue repair, by producing potential growth factors including hepatocyte growth factor (HGF). However, it remains elusive which factor activates macrophage in these processes. Intratracheal instillation of Pseudomonas aeruginosa caused neutrophil infiltration in the airspace; subsequently, the numbers of total AMs and neutrophil ingested AMs were increased. Bronchoalveolar lavage (BAL) fluid levels of monocyte chemoattractant protein (MCP)-1/CC chemokine ligand-2 (CCL2), a potent macrophage-activating factor, were increased before the increases in the number of AM ingesting neutrophils and HGF levels in BAL fluid. Immunoreactive MCP-1 proteins were detected in alveolar type II epithelial cells and AMs only after P. aeruginosa infection. The administration of anti-MCP-1/CCL2 Abs reduced the increases in the number of AM-ingesting neutrophils and HGF levels in BAL fluid, and eventually aggravated lung tissue injury. In contrast, the administration of MCP-1/CCL2 enhanced the increases in the number of AM ingesting neutrophils and HGF levels in BAL fluid, and eventually attenuated lung tissue injury. Furthermore, MCP-1/CCL2 enhanced the ingestion of apoptotic neutrophils and HGF production by a mouse macrophage cell line, RAW 267.4, in a dose-dependent manner. Collectively, MCP-1/CCL2 has a crucial role in the resolution and repair processes of acute bacterial pneumonia by enhancing the removal of dying neutrophils and HGF production by AMs.     

Role of insulin on PGE2 generation during LPS-induced lung inflammation in rats

Alterations in arachidonic acid (AA) metabolism have been reported to occur in diabetes mellitus. The present study was carried out to verify if these alterations are due to the relative lack of insulin or to high levels of blood glucose. Male Wistar rats were rendered diabetic by alloxan injection (42 mg/kg, i.v.), 10 or 30 days before the experiments. Some diabetic rats received a single dose (4 IU, s.c.) of NPH insulin 2 h before an intratracheal instillation of lipopolysaccharide (LPS, 750 microg) or saline. Six hours after LPS challenge, the following parameters were analysed: blood glucose levels, total and differential leukocyte counts in bronchoalveolar lavage (BAL) fluid; linoleic acid and AA content in blood neutrophils (HPLC), and levels of prostaglandin (PG)E(2) in BAL (ELISA). Relative to controls, a reduced number of neutrophils (18%) and decreased amounts of PGE(2) (40%) were observed in the BAL fluid of diabetic rats in response to LPS. A single dose of insulin was not able to reduce blood sugar levels to normal values, but instead resulted in the normalization of both leukocyte migration to the lungs and levels of PGE(2). Accordingly, these abnormalities might be primarily linked to a continuing insulin deficiency rather than to secondary hyperglycaemia occurring in the diabetic rat. In conclusion, data presented suggest that insulin might regulate neutrophil migration and generation of PGE(2) during the course of acute lung injury induced by LPS.     

Anti-inflammatory effect of pre-elafin in lipopolysaccharide-induced acute lung inflammation

The aim of the present study was to evaluate the anti-inflammatory activity of pre-elafin, an elastase-specific inhibitor, in lipopolysaccharide (LPS)-induced acute lung inflammation. C57BL/6 mice were pre-treated intranasally with recombinant human pre-elafin or vehicle only. One hour later, they were instilled intranasally with LPS (2 microg/mouse). Animals were sacrificed 6 hours after LPS instillation and bronchoalveolar lavage (BAL) was performed with three 1-ml aliquots of saline. LPS induced a lung inflammation characterised by a 100-fold increase in BAL neutrophils compared to control animals (265.8 +/- 54.5 x 10(3) and 2.4 +/- 1.3 x 10(3) neutrophils/ml, respectively). Pre-elafin dose-dependently reduced the neutrophil influx in the lung alveolar spaces by up to 84%. No elastase activity was detectable in all BAL fluids tested. Pre-elafin also reduced significantly LPS-induced gelatinase activity, as shown by zymography, and BAL macrophage inflammatory protein-2 (MIP-2) and KC levels, two potent neutrophil attractants and activators. Moreover, pre-elafin also significantly reduced mRNA levels of the three members of the IL-1 ligand family, namely IL-1alpha, IL-1beta and IL-1 receptor antagonist (IL-1Ra), type II IL-1 receptor, and TNFalpha as assessed in whole lung tissue by RNase protection assay. Thus, pre-elafin may be considered as a potent anti-inflammatory mediator.     

TGF-beta and Smad3 signaling link inflammation to chronic fibrogenesis

Transient adenovirus-mediated gene transfer of IL-1beta (AdIL-1beta), a proinflammatory cytokine, induces marked inflammation and severe and progressive fibrosis in rat lungs. This is associated with an increase in TGF-beta1 concentration in bronchoalveolar lavage (BAL) fluid. TGF-beta1 is a key cytokine in the process of fibrogenesis, using intracellular signaling pathways involving Smad2 and Smad3. In this study we investigate whether inflammation induced by IL-1beta is able to independently induce lung fibrosis in mice deficient in the Smad3 gene. Seven days after AdIL-1beta administration, similar levels of IL-1beta transgene are seen in BAL in both wild-type (WT) and knockout (KO) mice, and BAL cell profiles demonstrated a similar marked neutrophilic inflammation. Phospho-Smad2 staining was positive in areas of inflammation in both WT and KO mice at day 7. By day 35 after transient IL-1beta expression, WT mice showed marked fibrosis in peribronchial areas, quantified by picrosirius red staining and morphometry. However, there was no evidence of fibrosis or collagen accumulation in IL-1beta-treated KO mice, and peribronchial areas were not different from KO mice treated with the control adenovector. TGF-beta1 and phospho-Smad2 were strongly positive at day 35 in fibrotic areas observed in WT mice, but no such staining was detectable in KO mice. The IL-1beta-induced chronic fibrotic response in mouse lungs is dependent on Smad3. KO and WT animals demonstrated a similar inflammatory response to overexpression of IL-1beta indicating that inflammation must link to the Smad3 pathway, likely through TGF-beta, to induce progressive fibrosis.     

Carbon monoxide attenuates aeroallergen-induced inflammation in mice

Carbon monoxide (CO) generated by catalysis of heme by heme oxygenase is increased in the exhaled air of asthmatic patients. Based on recent studies demonstrating that asthma is an inflammatory disease associated with increased oxidants and that CO confers cytoprotection in oxidant-induced lung injury and inflammation, we sought to better understand the functional role of CO in asthma by using an aeroallergen model. Mice were sensitized to ovalbumin, challenged with aerosolized ovalbumin, and maintained in either CO (250 parts/million) or room air for 48 h. The differential effects of CO on bronchoalveolar lavage (BAL) fluid cell types were observed, with a marked attenuation of BAL fluid eosinophils in the CO-treated animals at 24 and 48 h. A marked reduction of the proinflammatory cytokine interleukin-5 was observed in the CO-treated mice, with no significant changes for other proinflammatory cytokines. These differential effects of CO were also observed with leukotrienes (LTs) and prostaglandins in that CO significantly decreased BAL fluid PGE2, and LTB4 but exerted negligible effect on thromboxane B2 or LTC4/D4/E4. Our data suggest a putative immunoregulatory role for CO in aeroallergen-induced inflammation in mice.     

Inhibition of tobacco smoke-induced lung inflammation by a catalytic antioxidant

Cigarette smokers experience airway inflammation and epithelial damage, the mechanisms of which are unknown. One potential cause may be free radicals either in tobacco smoke or produced during persistent inflammation. Inflammation may also be a driving force to cause airway epithelium to undergo changes leading to squamous cell metaplasia. To test whether tobacco smoke-induced inflammation could be reduced by a catalytic antioxidant, manganese(III)meso-tetrakis(N,N'-diethyl-1,3-imidazolium-2-yl) porphyrin (AEOL 10150) was given by intratracheal instillation to rats exposed to filtered air or tobacco smoke. Exposure to tobacco smoke for 2 d or 8 weeks (6 h/d, 3 d/week) significantly increased the number of cells recovered by bronchoalveolar lavage (BAL). AEOL 10150 significantly decreased BAL cell number in tobacco smoke-treated rats. Significant reductions in neutrophils were noted at 2 d and macrophages at 8 weeks. Lymphocytes were significantly reduced by AEOL 10150 at both time points. Squamous cell metaplasia following 8 weeks of tobacco smoke exposure was 12% of the total airway epithelial area in animals exposed to tobacco smoke without AEOL 10150, compared with 2% in animals exposed to tobacco smoke, but treated with AEOL 10150 (p <.05). We conclude that a synthetic catalytic antioxidant decreased the adverse effects of exposure to tobacco smoke.     

Intratracheal synthetic CpG oligodeoxynucleotide causes acute lung injury with systemic inflammatory response

Bacterial genome is characterized by frequent unmethylated cytosine-phosphate-guanine (CpG) motifs. Deleterious effects can occur when synthetic oligodeoxynucleotides (ODN) with unmethylated CpG dinucleotides (CpG-ODN) are administered in a systemic fashion. We aimed to evaluate the effect of intratracheal CpG-ODN on lung inflammation and systemic inflammatory response. C57BL/6J mice received intratracheal administration of CpG-ODN (0.01, 0.1, 1.0, 10, or 100 μM) or control ODN without CpG motif. Bronchoalveolar lavage (BAL) fluid was obtained 3 or 6 h or 1, 2, 7, or 14 days after the instillation and subjected to a differential cell count and cytokine measurement. Lung permeability was evaluated as the BAL fluid-to-plasma ratio of the concentration of human serum albumin that was injected 1 h before euthanasia. Nuclear factor (NF)-κB DNA binding activity was also evaluated in lung homogenates. Intratracheal administration of 10 μM or higher concentration of CpG-ODN induced significant inflammatory cell accumulation into the airspace. The peak accumulation of neutrophils and lymphocytes occurred 1 and 2 days after the CpG-ODN administration, respectively. Lung permeability was increased 1 day after the 10 μM CpG-ODN challenge. CpG-ODN also induced nuclear translocation of NF-κB and upregulation of various inflammatory cytokines in BAL fluid and plasma. Histopathology of the lungs and liver revealed acute lung injury and liver damage with necrosis, respectively. Control ODN without CpG motif did not induce any inflammatory change. Since intratracheal CpG-ODN induced acute lung injury as well as systemic inflammatory response, therapeutic strategies to neutralize bacterial DNA that is released after administration of bactericidal agents should be considered.     

Local nitric oxide levels reflect the degree of allergic airway inflammation after segmental allergen challenge in asthmatics.

Nitric oxide (NO) levels are increased in the exhaled air of asthmatics. As NO levels correlate with allergic airway inflammation, NO measurement has been suggested for disease monitoring. In patients with asthma, we previously demonstrated that intrabronchial treatment with a natural porcine surfactant enhanced airway inflammation after segmental allergen provocation. We studied whether local levels of NO reflect the degree of allergic airway inflammation following segmental allergen challenge with or without surfactant pretreatment. Segmental NO, as well as nitrite and nitrate in bronchoalveolar lavage (BAL) fluid, was measured before and after segmental challenge with either saline, saline plus allergen, or surfactant plus allergen in 16 patients with asthma and five healthy subjects. The data were compared with inflammatory BAL cells. Segmental NO levels were increased after instillation of saline (p < 0.05), or surfactant plus allergen in asthmatics (p < 0.05), and values were higher after surfactant plus allergen compared to saline challenge. Nitrate BAL levels were not altered after saline challenge but increased after allergen challenge (p < 0.05) and further raised by surfactant (p < 0.05), whereas nitrite levels were not altered by any treatment. Segmental NO and nitrate levels correlated with the degree of eosinophilic airway inflammation, and nitrate levels also correlated with neutrophil and lymphocyte numbers in BAL. In healthy subjects, NO, nitrite, and nitrate were unaffected. Thus, segmental NO and nitrate levels reflect the degree of allergic airway inflammation in patients with asthma. Measurement of both markers can be useful in studies using segmental allergen provocation, to assess local effects of potential immunomodulators.     

Effects of beta-adrenergic agonist infusions on myometrial activity and plasma prostaglandin levels in the nonpregnant sheep

Recent studies have reported that beta-adrenergic agonists stimulate the production of stimulatory prostaglandins (PGs) by intrauterine tissues in vitro. These drugs are used clinically to inhibit uterine contractions; consequently an increase in stimulatory PGs in vivo might have potentially adverse effects. We have, therefore, investigated whether beta-adrenergic agonists increase plasma PG concentrations in vivo. Samples of peripheral (aorta) and uterine venous enriched (vena cava) blood from nonpregnant sheep were collected at 15-min intervals for 1 h before, 3 h during, and 1 h postinfusion of either (a) the beta-adrenergic agonist isoproterenol (Isop) at a dose of 0.16 microgram.kg-1.min-1; (b) Isop at a dose of 0.08 microgram.kg-1.min-1; or (c) saline, 1 mL/h via a jugular vein catheter. The sheep were also equipped with intrauterine recording balloons to record intrauterine pressure and myometrial electromyographic (EMG) electrodes to measure EMG activity. Infusion of Isop at 0.16 microgram.kg-1.min-1 produced a significant initial inhibition of uterine activity, although contractions returned (within 60 min) despite continued administration of Isop. Plasma PGE2 (but not PGF2 alpha or 13,14-dihydro-15-keto-PGF2 alpha (PGFM] concentrations were significantly elevated during the Isop infusion. Administration of Isop at 0.08 microgram.kg-1.min-1 produced no effects on uterine contractile activity but was associated with a significant elevation in plasma PGE2 (but not PGF2 alpha or PGFM) concentrations. No changes in plasma PGE2, PGF2 alpha, or PGFM occurred during saline infusion.(ABSTRACT TRUNCATED AT 250 WORDS)     

RAGE signaling by alveolar macrophages influences tobacco smoke-induced inflammation

Receptors for advanced glycation end-products (RAGE) are multiligand cell surface receptors of the immunoglobin family expressed by epithelium and macrophages, and expression increases following exposure to cigarette smoke extract (CSE). The present study sought to characterize the proinflammatory contributions of RAGE expressed by alveolar macrophages (AMs) following CSE exposure. Acute exposure of mice to CSE via nasal instillation revealed diminished bronchoalveolar lavage (BAL) cellularity and fewer AMs in RAGE knockout (KO) mice compared with controls. Primary AMs were obtained from BAL, exposed to CSE in vitro, and analyzed. CSE significantly increased RAGE expression by wild-type AMs. Employing ELISAs, wild-type AMs exposed to CSE had increased levels of active Ras, a small GTPase that perpetuates proinflammatory signaling. Conversely, RAGE KO AMs had less Ras activation compared with wild-type AMs after exposure to CSE. In RAGE KO AMs, assessment of p38 MAPK and NF-κB, important intracellular signaling intermediates induced during an inflammatory response, revealed that CSE-induced inflammation may occur in part via RAGE signaling. Lastly, quantitative RT-PCR revealed that the expression of proinflammatory cytokines including TNF-α and IL-1β were detectably decreased in RAGE KO AMs exposed to CSE compared with CSE-exposed wild-type AMs. These results reveal that primary AMs orchestrate CSE-induced inflammation, at least in part, via RAGE-mediated mechanisms.     

Adiponectin-deficient mice are protected against tobacco-induced inflammation and increased emphysema

Adiponectin is a cytokine with both proinflammatory and anti-inflammatory properties that is expressed in epithelial cells in the airway in chronic obstructive pulmonary disease-emphysema (COPD-E). To determine whether adiponectin modulates levels of lung inflammation in tobacco smoke-induced COPD-E, we used a mouse model of COPD-E in which either adiponectin-deficient or wild-type (WT) mice were exposed to tobacco smoke for 6 mo. Outcomes associated with tobacco smoke-induced COPD-E were quantitated including lung inflammation [bronchoalveolar lavage (BAL) and total and differential cell count], lung mediators of inflammation (cytokines and chemokines), air space enlargement (i.e., linear intercept), and lung function (tissue elastance) in the different groups of mice. Whereas exposure of WT mice to tobacco smoke for 6 mo induced significant lung inflammation (increased total BAL cells, neutrophils, and macrophages), adiponectin-deficient mice had minimal BAL inflammation when exposed to tobacco smoke for 6 mo. In addition, whereas chronic tobacco-exposed WT mice had significantly increased levels of lung mediators of inflammation [i.e., TNF-α, keratinocyte-derived chemokine (KC), and adiponectin] as well as significantly increased air space enlargement (increased linear intercept) and decreased tissue elastance, exposure of adiponectin-deficient mice to chronic tobacco smoke resulted in no further increase in lung mediators, air space enlargement, or tissue elastance. In vitro studies demonstrated that BAL macrophages derived from adiponectin-deficient mice incubated in media containing tobacco smoke expressed minimal TNF-α or KC compared with BAL macrophages from WT mice. These studies suggest that adiponectin plays an important proinflammatory role in tobacco smoke-induced COPD-E.     

Alveolar macrophages from systemic sclerosis patients: evidence for IL-4-mediated phenotype changes

The mechanism of chronic lung inflammation leading to lung fibrosis is unknown and does not have a characteristic inflammatory macrophage phenotype. This study was undertaken to determine whether a change in macrophage phenotype could account for chronic lung inflammation. In this study, human alveolar macrophages (AM) from subjects with systemic sclerosis (SSc) were obtained from bronchoalveolar lavage (BAL) and characterized on the basis of function (response to LPS), phenotype, and relative cell-surface B7 expression. AM from the subjects' disease-involved and noninvolved lung lobes were compared with each other and to AM from normal volunteer BAL. AM from involved SSc lobes produced significantly more interleukin (IL)-1beta and PGE(2) than AM from uninvolved lobes in response to LPS, but there was no spontaneous production of either mediator. The activator AM phenotype designated by RFD1+ surface epitope was significantly elevated in SSc BAL samples compared with normal BAL, although there were no differences comparing involved vs. noninvolved lobes within SSc subjects. The major histocompatibility complex II costimulatory molecule B7.2 was also significantly elevated in SSc AM compared with normal AM, again with no differences between involved and noninvolved lobes. In an attempt to determine environmental influences on AM phenotypes, normal AM were cultured in vitro with IFN-gamma, IL-3, IL-4, IL-10, IL-12, or dexamethasone for 6 days. Of the cytokines examined, only IL-4 induced significant increases in both the activator phenotype RFD1+ and B7.2 expression. Taken together, these results indicate that IL-4 could account for proinflammatory AM phenotype changes and B7 surface-marker shifts, as seen in subjects with SSc.     

Immunohistological characterization of leukocytes in the lungs of healthy mice and after bacterial intratracheal infection

Leukocytes in the peripheral lung parenchyma of mice have not been characterized histologically during bacterial infection. The aim of this study was to investigate (a) the immunohistological characteristics of healthy murine lungs and (b) the cell kinetics during acute inflammation. BALB/c and MF1 mice were examined; as well as transgenic mice with the gene defect of cystic fibrosis (CF) in the airways as an animal model for this disease. MF1 mice served as controls for the transgenic animals. Lavaged and perfused lungs were snap frozen. B and T lymphocytes, CD4+ and CD8+ cells, dendritic cells, neutrophils and a subset of macrophages were enumerated on cryostat lung sections. The lung tissue and bronchoalveolar lavage (BAL) of BALB/c mice, infected intratracheally with Haemophilus influenzae type b (Hib), were studied at different time points after infection. In the lungs of healthy mice, including CF mice, the largest population was that of T cells, CD4+ cells being always more frequent than CD8+ cells. During acute inflammation the number of neutrophils in the lung parenchyma and BAL increased strongly within the first hours after bacterial instillation and reached baseline levels within one week. This study provides a semi-quantitative analysis of immunocompetent cells in normal and infected murine lung tissue. Differences in cell numbers are found between different strains. Moreover, the cellular reaction during Hib infection in mouse lungs is dominated by neutrophils, as expected in a primary immune response. In uninfected CF mice the numbers and distribution of immune cells in the lung tissue are normal, indicating that the cellular defense is adequate.     

Role of CXCR2 in cigarette smoke-induced lung inflammation

It has been hypothesized that the destruction of lung tissue observed in smokers with chronic obstructive pulmonary disease and emphysema is mediated by neutrophils recruited to the lungs by smoke exposure. This study investigated the role of the chemokine receptor CXCR2 in mediating neutrophilic inflammation in the lungs of mice acutely exposed to cigarette smoke. Exposure to dilute mainstream cigarette smoke for 1 h, twice per day for 3 days, induced acute inflammation in the lungs of C57BL/6 mice, with increased neutrophils and the neutrophil chemotactic CXC chemokines macrophage inflammatory protein (MIP)-2 and KC. Treatment with SCH-N, an orally active small molecule inhibitor of CXCR2, reduced the influx of neutrophils into the bronchoalveolar lavage (BAL) fluid. Histological changes were seen, with drug treatment reducing perivascular inflammation and the number of tissue neutrophils. beta-Glucuronidase activity was reduced in the BAL fluid of mice treated with SCH-N, indicating that the reduction in neutrophils was associated with a reduction in tissue damaging enzymes. Interestingly, whereas MIP-2 and KC were significantly elevated in the BAL fluid of smoke exposed mice, they were further elevated in mice exposed to smoke and treated with drug. The increase in MIP-2 and KC with drug treatment may be due to the decrease in lung neutrophils that either are not present to bind these chemokines or fail to provide a feedback signal to other cells producing these chemokines. Overall, these results demonstrate that inhibiting CXCR2 reduces neutrophilic inflammation and associated lung tissue damage due to acute cigarette smoke exposure.     

LPS-induced lung inflammation in marmoset monkeys - an acute model for anti-inflammatory drug testing

Increasing incidence and substantial morbidity and mortality of respiratory diseases requires the development of new human-specific anti-inflammatory and disease-modifying therapeutics. Therefore, new predictive animal models that closely reflect human lung pathology are needed. In the current study, a tiered acute lipopolysaccharide (LPS)-induced inflammation model was established in marmoset monkeys (Callithrix jacchus) to reflect crucial features of inflammatory lung diseases. Firstly, in an ex vivo approach marmoset and, for the purposes of comparison, human precision-cut lung slices (PCLS) were stimulated with LPS in the presence or absence of the phosphodiesterase-4 (PDE4) inhibitor roflumilast. Pro-inflammatory cytokines including tumor necrosis factor-alpha (TNF-α) and macrophage inflammatory protein-1 beta (MIP-1β) were measured. The corticosteroid dexamethasone was used as treatment control. Secondly, in an in vivo approach marmosets were pre-treated with roflumilast or dexamethasone and unilaterally challenged with LPS. Ipsilateral bronchoalveolar lavage (BAL) was conducted 18 hours after LPS challenge. BAL fluid was processed and analyzed for neutrophils, TNF-α, and MIP-1β. TNF-α release in marmoset PCLS correlated significantly with human PCLS. Roflumilast treatment significantly reduced TNF-α secretion ex vivo in both species, with comparable half maximal inhibitory concentration (IC(50)). LPS instillation into marmoset lungs caused a profound inflammation as shown by neutrophilic influx and increased TNF-α and MIP-1β levels in BAL fluid. This inflammatory response was significantly suppressed by roflumilast and dexamethasone. The close similarity of marmoset and human lungs regarding LPS-induced inflammation and the significant anti-inflammatory effect of approved pharmaceuticals assess the suitability of marmoset monkeys to serve as a promising model for studying anti-inflammatory drugs.     

Alanyl-glutamine resolves lipopolysaccharide-induced lung injury in mice by modulating the polarization of regulatory T cells and T helper 17 cells

Regulatory T cells (Tregs) and T helper (Th) 17 cells are two subsets of Th cells with opposing actions which participate in the process of resolving acute lung injury (ALI). Glutamine (Gln) is a nutrient commonly used in nutrition regimens due to its immunomodulatory effects. This study investigated whether alanyl-glutamine (Ala-Gln) administration modulated polarization of Th subsets in a model of lipopolysaccharide (LPS)-induced ALI during the resolution period. Intratracheal instillation of LPS in male C57BL/6J mice was used to induce ALI. On day 1 after LPS instillation, mice in the Gln group were given an intragastric gavage of 0.75 g Ala-Gln/kg daily, whereas the control (Con) group received identical amounts of sterilized distilled water. At 4, 7 and 10 days after a single dose of LPS, mice were killed by cardiac puncture. Bronchoalveolar lavage (BAL) fluid and lung tissues were collected for further analysis. Compared to the Con group, weight loss was less in the Gln group. Percentages of Tregs and interleukin-2 levels in BAL fluid increased, whereas Th17 cells were suppressed in the Gln group. Neutrophil clearance was promoted in the Gln group. Expressions of proinflammatory-related and fibrosis-related genes in lung tissues decreased in the Gln group. Histopathological findings also showed that interstitial inflammation was less severe in the Gln group. These results suggest that Ala-Gln administration after the onset of ALI can help resolve lung inflammation and injury by modulating the polarization of Tregs and Th17 cells.     

PGE1 inhibited PMN attachment to air emboli in vivo during infusion of ZAP without preventing lung injury.

Prostaglandin E1 (PGE1) treatment of neutrophils inhibits their adherence to substrates in vitro, including endothelial cell monolayers. Demonstration that PGE1 inhibits neutrophil adherence in vivo in the lung, however, is complicated by PGE1 effects on cells other than neutrophils, such as endothelial cells. To determine whether PGE1 inhibits neutrophil adherence properties in vivo, we used air emboli as intravascular targets for neutrophil attachment. Four experimental conditions were studied in anesthetized and awake sheep that were treated with 1) PGE1 and air emboli, 2) saline and air emboli, 3) PGE1 and zymosan-activated plasma (ZAP) + air emboli, and 4) saline and ZAP + air emboli. PGE1 (30 ng.kg-1.min-1) or saline was infused continuously 1 h before and 1 h during the infusion of air emboli (group 1; n = 13 sheep) or ZAP + air emboli (group 2; n = 13 sheep). The number of neutrophils (PMNs) attached to air emboli in four anesthetized sheep per condition was significantly less in sheep given PGE1 and ZAP + air emboli [8 +/- 3 (SD) PMNs/mm of embolus perimeter] than in the other three conditions (14-21 PMNs/mm; P less than 0.05). Repeated experiments in five awake sheep per group showed that PGE1 treatment did not prevent increased lung lymph protein clearance in either group compared with saline treatment. We conclude that PGE1 specifically inhibited attachment of ZAP-activated neutrophils to air emboli in vivo. The lack of pathophysiological protection suggests that PGE1-induced alterations in neutrophil attachment properties were independent of other cellular activation responses.