p47phox and reactive oxygen species production modulate expression of microRNA-451 in macrophages.

The production of microRNAs (miRNA) is influenced by various stimuli, including environmental stresses. We hypothesized that reactive oxygen species (ROS)-associated stress could regulate macrophage miRNA synthesis. miRNAs undergo unique steps of maturation processing through either one of two pathways of cytoplasmic processing. Unlike the canonical pathway, the regulation of alternative cytoplasmic processing of miRNA has not been fully elucidated yet. We cultured bone marrow derived macrophages (BMDM) from wild type (WT) and p47(phox-/-) mice and profiled miRNA expression using microarrays. We analyzed 375 miRNAs including four endogenous controls to normalize the data. At resting state, p47(phox-/-) BMDM has the markedly reduced expression of miR-451 compared to WT BMDM, without other significant differences. Unlike majority of miRNAs, miR-451 goes through the unique alternative processing pathway, in which Ago2 plays a key role. In spite of significant reduction of mature miR-451, however, its precursor form, pre-mir-451, was similar in both BMDMs, suggesting that the processing of pre-mir-451 is impaired in p47(phox-/-) BMDM. Moreover, p47(phox-/-) BMDM expressed significantly reduced level of Ago2. In contrast, Ago2 mRNA levels were similar in WT and p47(phox-/-) BMDM, suggesting a post-transcriptional defect of Ago2 production in p47(phox-/-) macrophages, which resulted in impaired processing of pre-miR-451. In order to examine the functional significance of miR-451 in macrophages, we cultured BMDMs from miR-451 knock-out mice. Of interest, miR-451-deficient BMDM exhibited reduced ROS generation upon zymosan stimulation, compared to WT BMDM. Our studies suggest functional crosstalk between ROS and miR-451 in the regulation of macrophage oxidant stress.

Expression of TREM-1 is inhibited by PGD2 and PGJ2 in macrophages.

TREM-1 is a superimmunoglobulin receptor present on neutrophils and monocytes, which plays an important role in the amplification of inflammation. The natural ligands for TREM-1 have not been identified; however, Toll-like receptor ligands are known to induce the expression of TREM-1. Blockade of TREM-1 has shown to improve survival in animal models of sepsis. In the present studies, we investigated the role of lipid mediators in the expression of TREM-1. In a macrophage cell line, we show that the expression of TREM-1 in response to LPS and bacteria Pseudomonas aeruginosa is inhibited by PGD(2) and cyclopentanone prostaglandins PGJ(2) and 15-dPGJ(2). The inhibition of TREM-1 by these prostaglandins is independent of the PGD(2) receptors and PPARγ but occurs by activation of Nrf2 and inhibition of NF-κB. Our data suggest a novel mechanism by which these prostaglandins exhibit anti-inflammatory effects and a new therapeutic approach to inhibition of TREM-1.

Cigarette smoking and innate immunity.

Cigarette smoking is a worldwide epidemic and the most prevalent cause of many diseases leading to increased morbidity and mortality globally. The impact of smoking on pathogenesis of cancer is being extensively studied however cigarette smoke as an immunosuppressant is less well recognized. Here we review the immunosuppressive effects of cigarette smoke and the mechanisms by which smoking affects host innate immunity including structural and functional changes in the respiratory ciliary epithelium, lung surfactant protein, and immune cells such as alveolar macrophages, neutrophils, lymphocytes and natural killer (NK) cells. Thus smoking cessation should be emphasized not only for prevention of cancer and coronary artery disease but also for patients with recurrent infections and immunosuppressive states.

The nuclear factor kappa-B pathway in airway epithelium regulates neutrophil recruitment and host defence following Pseudomonas aeruginosa infection.

Pseudomonas aeruginosa pneumonia usually results from a deficit of the innate immune system. To investigate whether inflammatory signalling by airway epithelial cells provides a pivotal line of defence against P. aeruginosa infection, we utilized two separate lines of inducible transgenic mice that express a constitutive activator of the nuclear factor kappa-B (NF-kappaB) pathway (IKTA) or a dominant inhibitor of NF-kappaB (DNTA) in airway epithelial cells. Compared with control mice, IKTA mice showed an enhanced host response to P. aeruginosa infection with greater neutrophil influx into the lungs, increased expression of Glu-Leu-Arg-positive (ELR(+)) CXC chemokines macrophage inflammatory protein-2 and keratinocyte chemoattractant (KC), superior bacterial clearance and improved survival at 24 h after infection. Neutrophil depletion abrogated the improvement in host defence identified in IKTA mice. In contrast, DNTA mice showed impaired responses to P. aeruginosa infection with higher bacterial colony counts in the lungs, decreased neutrophilic lung inflammation and lower levels of KC in lung lavage fluid. DNTA mice given recombinant KC at the time of P. aeruginosa infection demonstrated improved neutrophil recruitment to the lungs and enhanced bacterial clearance. Our data indicate that the NF-kappaB pathway in airway epithelial cells plays an essential role in defence against P. aeruginosa through generation of CXC chemokines and recruitment of neutrophils.

Functional genomics of silencing TREM-1 on TLR4 signaling in macrophages.

Triggering receptor expressed on myeloid cells 1 (TREM-1) is a recently discovered molecule that is expressed on the cell surface of monocytes and neutrophils. Engagement of TREM-1 triggers synthesis of proinflammatory cytokines in response to microbes, but the extent and mechanism by which TREM-1 modulates the inflammatory response is poorly defined. In the present study, we investigated the functional effects of blocking TREM-1 on the Toll-like receptor (TLR)4-mediated signaling pathway in macrophages. By transfecting cells with small hairpin interfering RNA molecules to TREM-1 (shRNA), we confirmed that TREM-1 mRNA and protein expression was greatly attenuated in RAW cells in response to treatment with LPS. PCR array for genes related to or activated by the TLR pathway revealed that although the expression of TLR4 itself was not significantly altered by silencing of TREM-1, expression of several genes, including MyD88, CD14, IkappaBalpha, IL-1beta, MCP-1, and IL-10 was significantly attenuated in the TREM-1 knockdown cells in response to treatment with LPS. These data indicate that expression of TREM-1 modulates the TLR signaling in macrophages by altering the expression of both adaptor and effector proteins that are critical to the endotoxin response.

Conditional regulation of cyclooxygenase-2 in tracheobronchial epithelial cells modulates pulmonary immunity.

Cyclooxygenase-2 (COX-2) gene expression in the lung is induced in pathological conditions such as asthma and pneumonia; however, the exact impact of COX-2 gene expression in the airway in regulating inflammatory and immunological response in the lung is not understood. To define a physiological role of inducible COX-2 in airway epithelial cells, we developed a novel line of transgenic mice, referred to as CycloOxygenase-2 TransActivated (COTA) mice, that overexpress a COX-2 transgene in the distribution of the CC-10 promoter in response to doxycycline. In response to doxycycline treatment, COX-2 expression was increased in airway epithelium of COTA mice and whole lung tissue contained a three- to sevenfold increase in prostaglandin E(2) (PGE(2)), prostaglandin D(2) (PGD(2)) thromboxane B(2) (TXB(2)) and 6-Keto prostaglandin F(2alpha) (PGF(2alpha)) compared to wild-type and untreated COTA mice. Interestingly, primary mouse tracheal epithelial cells from COTA mice produced only PGE(2) by doxycycline-induced COX-2 activation, providing an indication of cellular specificity in terms of mediator production. In the ovalbumin model, in which doxycycline was given at the sensitization stage, there was an increase in interleukin (IL)-4 level in lung tissue from COTA mice compared to untreated COTA and wild-type mice. In addition, COTA mice that were treated with doxycycline had impaired clearance of Pseudomonas aeruginosa pneumonia compared to wild-type mice. COX-2 gene expression in airway epithelial cells has an important role in determining immunological response to infectious and allergic agents.

Molecular targets for modulating lung inflammation and injury.

The inflammatory response of the lung and airways is one of the main targets for tile development of new therapies for variety of disorders including the acute respiratory distress syndrome, cystic fibrosis, idiopathic pulmonary fibrosis, and chronic obstructive pulmonary disease. Over the last decade our understanding of the molecular biology of the inflammatory response has advanced considerably and has opened up new avenues for therapeutic intervention. Furthermore, the mechanism of action of many of the existing anti-inflammatory agents has been revealed by this burgeoning information. Here, we discuss the functions and therapeutic potential of molecules that might prove promising as targets for treatment of inflammatory lung diseases. These possible molecular targets include cell surface proteins/receptors [toll like receptors (TLRs), triggering receptors expressed on myeloid cells (TREMs), and syndecans)], transcription factors [NF-kappaB, AP-1, PU.1, and high mobility group box 1 (HMGB1)], and regulatory proteins [macrophage migration inhibitory factor (MIF), granulocyte macrophage colony stimulating factor (GM-CSF), cyclooxygenase 2 (COX-2), heme oxygenase 1 (HO-1)].

High-dose dexamethasone accentuates nuclear factor-kappa b activation in endotoxin-treated mice.

We examined the effects of dexamethasone treatment on nuclear factor (NF)-kappa B activation and lung inflammation in transgenic reporter mice expressing photinus luciferase under the control of an NF-kappa B-dependent promoter (HLL mice). In vitro studies with bone marrow and peritoneal macrophages derived from these mice showed that treatment with dexamethasone blocked luciferase induction after treatment with Escherichia coli lipopolysaccharide (LPS); however, treatment of mice with intraperitoneal injection of dexamethasone at doses of 0.3 microg/g and 1 microg/g failed to inhibit NF-kappa B-dependent luciferase activity in the lungs. Furthermore, intraperitoneal treatment with 10 microg/g of dexamethasone prior to LPS paradoxically resulted in augmented luciferase activity as compared with that of mice treated with LPS alone. NF-kappa B-dependent luciferase expression in the lungs was detected by bioluminescence imaging and by measurement of luciferase activity in homogenized lung tissue. In these studies, there was an excellent correlation between indirect measurement of luciferase activity by bioluminescence in living mice and direct measurement of luciferase activity in lung tissue. Dexamethasone treatment did not affect LPS-induced neutrophilic influx or the concentration of macrophage inflammatory protein-2 in lung lavage fluid. These findings emphasize the potential error of extrapolating in vitro findings to complex in vivo events such as regulation of inflammation.

Sarcoidosis and opportunistic infections.

Two patients receiving steroid therapy for sarcoidosis had a potentially fatal opportunistic infection that was difficult to differentiate from the underlying illness, but was successfully treated after the diagnosis was made. The effects of sarcoidosis on the immune system and the additional effects of steroid therapy on cell-mediated immunity seem to be of real clinical significance, rather than being of theoretical interest alone, because of the risk of infection with intracellular infecting organisms, even if such infections occur relatively infrequently.

Pleural fluid characteristics of patients with symptomatic pleural effusion after coronary artery bypass graft surgery.

BACKGROUND: This study describes the pleural fluid characteristics of patients who develop symptomatic pleural effusions after coronary artery bypass graft surgery (CABG). METHODS: Post-CABG patients who underwent a therapeutic thoracentesis for a symptomatic pleural effusion were included unless another explanation for the pleural effusion was present. RESULTS: During the study, 71 patients (mean age, 61 years) were identified; 49 were men and 22 were women. All patients underwent internal mammary artery grafting. Early effusions (<30 days after CABG) occurred in 45 patients (63%) and late effusions (>/=30 days after CABG) developed in 26 (37%). Early effusions were bloody (median red blood cell count, 706 x 10(12)/L [706,000 mm(3)])with a high eosinophil count (median, 0.385), whereas effusions that occurred in the late period were yellow exudates with predominant lymphocytes (median, 0.68) and monocytes (median, 0.20). The mean pleural fluid level of lactate dehydrogenase was more than 3 times the upper limit of the reference range in serum in early effusions, whereas late effusions had significantly lower lactate dehydrogenase levels. CONCLUSIONS: Characteristics of early and late effusions differ significantly, suggesting a different pathogenesis of the effusions. Patients who develop a symptomatic pleural effusion after CABG should undergo a therapeutic thoracentesis; however, further investigations are warranted only in patients who have pleural fluid characteristics different from those described.

The role of nuclear factor-kappa B in pulmonary diseases.

Nuclear factor-kappa B (NF-kappaB) is a family of DNA-binding protein factors that are required for transcription of most proinflammatory molecules, including adhesion molecules, enzymes, cytokines, and chemokines. NF-kappaB activation seems to be a key early event in a variety of cell and animal model systems developed to elucidate the pathobiology of lung diseases. The purpose of this short review is to describe what is known about the molecular biology of NF-kappaB and to review information that implicates NF-kappaB in the pathogenesis of lung disease, including ARDS, systemic inflammatory response syndrome, asthma, respiratory viral infections, occupational and environmental lung disease, and cystic fibrosis.

Respiratory bronchiolitis associated with severe dyspnea, exertional hypoxemia, and clubbing.

Respiratory bronchiolitis-associated interstitial lung disease (RBILD) is a distinct clinicopathologic disease described almost exclusively in cigarette smokers.(1) (2) The disease usually presents with mild symptoms and is associated with a good prognosis. (2) Severe lung dysfunction has not been reported with RBILD, which is often confused clinically and radiographically with desquamative interstitial lung disease or idiopathic pulmonary fibrosis (IPF). Two patients with RBILD who developed severe dyspnea, hypoxemia, and clubbing are described. Initially, IPF was diagnosed in both patients. The severity of symptoms was such that the first patient's room air saturation was 85% and the second patient had severe impairment of lung function, with FEV(1) of 39% and FVC of 40%. Advanced lung disease required supplemental home oxygen therapy in the first patient and referral for lung transplant evaluation in the second patient. After a detailed review of histology revealed a diagnosis of RBILD, both patients were encouraged to stop smoking; smoking cessation led to considerable improvement in symptoms and lung function tests. We conclude that advanced lung dysfunction occurs in some patients with RBILD and should not dissuade that diagnosis.

Breast metastasis from non-small cell lung cancer.

Breast is an unusual site for metastatic disease, particularly for non-small cell lung cancer. We report an unusual case of metastatic breast lesions from a primary anaplastic lung tumor and discuss the common and uncommon sites of metastasis from lung carcinomas.

Recurrence of primary spontaneous pneumothorax.

BACKGROUND: Primary spontaneous pneumothorax (PSP) is a common clinical problem and its incidence is thought to be increasing. The risk of recurrence is high and various studies quote rates of 20-60%. Factors which may or may not predispose to recurrence have not yet been established. METHODS: In a study period of four years 291 cases with a diagnosis of pneumothorax were reviewed; 153 patients with PSP were included in the study. Their risk of recurrence was analysed with particular reference to the following variables: age, sex, height and body mass index (BMI) of the patient, the initial size of pneumothorax, the smoking status of the patient, and the primary form of treatment employed. Univariate analysis was carried out by chi 2 testing and multivariate analysis was calculated by a logistic regression model. RESULTS: A retrospective study of 275 episodes of PSP in 153 patients over a four year period confirmed a high incidence of recurrence (54.2%). PSP was twice as common in men as in women, though women were significantly more likely to develop a recurrence (chi 2 = 7.58, df = 1, p < 0.01). Male height was the second most important factor, and smoking cessation the only other variable which significantly influenced the risk of recurrence. CONCLUSIONS: Analysis of several potential risk factors revealed that recurrence was not related to the BMI of the patient, the initial treatment of the pneumothorax, nor to its size. Recurrence was more common in taller men and in women. Smoking cessation appeared to reduce the risk of recurrence. These findings are discussed in the context of the possible aetiology of spontaneous pneumothorax, recurrences, and the management thereof.

Spontaneous pneumothorax in a patient with an azygos lobe.

The association between a spontaneous pneumothorax and an azygos lobe is surprisingly rare. A case is reported in which surgical management was difficult; it is suggested that thoracotomy is preferable to video-assisted thoracoscopic surgery in this situation. It is possible that the presence of an azygos lobe might protect against the subsequent development of a spontaneous pneumothorax, and the possible mechanism of this is discussed.

Recurrent respiratory papillomatosis with pulmonary cystic disease in a child, following maternal genital warts.

Human papillomavirus (HPV) infection of the genital tract is associated with the development of genital warts. A causal link between maternal HPV infection and development of laryngeal papillomatosis in the offspring has been proposed. We report a case of pulmonary cystic disease, a rare but serious complication of laryngeal papillomatosis in a child, following maternal genital warts.