Activation of innate immunity in the lungs can lead to a self-limited inflammatory response or progress to severe lung injury. We investigated whether specific parameters of NF-κB pathway activation determine the outcome of acute lung inflammation using a novel line of transgenic reporter mice. Following a single i.p. injection of Escherichia coli LPS, transient NF-κB activation was identified in a variety of lung cell types, and neutrophilic inflammation resolved without substantial tissue injury. However, administration of LPS over 24 h by osmotic pump (LPS pump) implanted into the peritoneum resulted in sustained, widespread NF-κB activation and neutrophilic inflammation that culminated in lung injury at 48 h. To determine whether intervention in the NF-κB pathway could prevent progression to lung injury in the LPS pump model, we administered a specific IκB kinase inhibitor (BMS-345541) to down-regulate NF-κB activation following the onset of inflammation. Treatment with BMS-345541 beginning at 20 h after osmotic pump implantation reduced lung NF-κB activation, concentration of KC and MIP-2 in lung lavage, neutrophil influx, and lung edema measured at 48 h. Therefore, sustained NF-κB activation correlates with severity of lung injury, and interdiction in the NF-κB pathway is beneficial even after the onset of lung inflammation.

Malignancy involving the pleura is the third leading cause of pleural effusions, with an annual incidence of Ͼ 150,000 cases in the United States (1, 2). Adenocarcinomas account for ‫ف‬ 70% of all malignant pleural effusions (MPEs), with lung adenocarcinomas being the most frequent underlying malignancy (3). The appearance of a MPE is an ominous prognostic sign for patients with cancer, because the presence of the MPE indicates that the tumor is incurable by surgery and life expectancy is short (1). In addition, the presence of the pleural effusion can cause dyspnea that severely compromises the quality of the patient's life (4, 5). Pleurodesis, the iatrogenic induction of pleural fibrosis to obliterate the pleural cavity, is commonly used to prevent symptomatic re-accumulation of pleural effusions; however, this therapy is often ineffective and is associated with significant It is generally believed that disruption of the endothelialmesothelial barrier, increased capillary permeability, tumorinduced angiogenesis, and lymphatic obstruction are responsible for the exudation of increased amounts of fluid into the pleural cavity (1, 7). However, the specific mechanisms underlying pleural fluid accumulation are poorly defined because studies of MPE pathogenesis are limited by a lack of animal models that reproduce the pathobiology of human MPE. Although mouse models that require immunocompromised mice for propagation of human cancer cells mice have provided insights into the biological behavior of tumor cells in the pleural cavity (7-9), these models are not ideal because the host is immunocompromised, and, therefore, the host immune response is attenuated or missing. The immune response against tumor may be an important component in the development of MPE because host inflammatory cells may contribute to or regulate the production of mediators that affect pathogenesis (10).Nuclear factor (NF)-B is a ubiquitous family of transcription regulatory proteins that affects a variety of cellular functions and influences tumor biology and host-tumor interactions. NF-B is activated by a number of tumor-promoting agents and is involved in the production of proteins that enhance cell survival and proliferation (11). High basal NF-B activation is present in lung adenocarcinoma cells and human lung cancer, and inhibition of NF-B sensitizes tumor cells to apoptosis and the effects of chemotherapeutic agents (11)(12)(13)(14)(15).Lewis lung cancer (LLC) cells are derived from a spontaneously arising lung adenocarcinoma in C57B/6 mice. These cells are characterized by short doubling times in vitro and in vivo and aggressive biological behavior. They can be propagated in wild-type C57B/6 mice, giving rise to lung adenocarcinomas (16)(17)(18). In these studies, we have developed and characterized a new murine model of MPE after instillation of lung adenocarcinoma cells in the pleural space of immunocompetent mice. We used this model to investigate whether the NF-B pathway in tumor cells is linked to MPE formation and progression of pl...

We investigated the impact of inflammatory signaling in airway epithelial cells on host defense against Pseudomonas aeruginosa, a major cause of nosocomial pneumonia. In mice, airway instillation of P. aeruginosa resulted in NF-κB activation in the lungs that was primarily localized to the bronchial epithelium at 4 h, but was present in a variety of cell types by 24 h. We modulated NF-κB activity in airway epithelium by intratracheal delivery of adenoviral vectors expressing RelA (AdRelA) or a dominant inhibitor of NF-κB before P. aeruginosa infection. Bacterial clearance was enhanced by up-regulation of NF-κB activity following AdRelA administration and was impaired by treatment with a dominant inhibitor of NF-κB. The TNF-α concentration in lung lavage was increased by AdRelA treatment and beneficial effects of NF-κB up-regulation were abrogated in TNF-α-deficient mice. In contrast, NF-κB inhibition reduced MIP-2 expression and neutrophil influx following P. aeruginosa infection. Therefore, inflammatory signaling through the NF-κB pathway in airway epithelial cells critically regulates the innate immune response to P. aeruginosa.

To determine whether NF-κB activation is sufficient to generate lung inflammation in vivo, we selectively expressed a constitutively active form of IκB kinase 1 (cIKK1) or IκB kinase 2 (cIKK2) in airway epithelium. After intratracheal administration of adenoviral vectors expressing cIKK1 or cIKK2 to transgenic reporter mice that express Photinus luciferase under the control of an NF-κB-dependent promoter, we detected significantly increased luciferase activity over time (up to 96 h). Compared with control mice treated with adenoviral vectors expressing β-galactosidase, lung bioluminescence and tissue luciferase activity were increased in NF-κB reporter mice treated with adenovirus (Ad)-cIKK1 or Ad-cIKK2. NF-κB activation in lungs of Ad-cIKK1- and Ad-cIKK2-treated mice was confirmed by immunoblots for RelA and EMSA from lung nuclear protein extracts. Mice treated with Ad-cIKK1 or Ad-cIKK2 showed induction of mRNA expression of several chemokines and cytokines in lung tissue. In lung lavage fluid, mice treated with Ad-cIKK1 or Ad-cIKK2 showed elevated concentrations of NF-κB-dependent chemokines macrophage-inflammatory protein 2 and KC and increased numbers of neutrophils. Coadministration of adenoviral vectors expressing a transdominant inhibitor of NF-κB with Ad-cIKK1 or Ad-cIKK2 resulted in abrogated NF-κB activation and other parameters of lung inflammation, demonstrating that the observed inflammatory effects of Ad-cIKK1 and Ad-cIKK2 were dependent on NF-κB activation by these kinases. These data show that selective expression of IκB kinases in airway epithelium results in NF-κB activation, inflammatory mediator production, and neutrophilic lung inflammation. Therapies targeted to NF-κB in lung epithelium may be beneficial in treating inflammatory lung diseases.

Although both inflammation and oxidative stress contribute to the pathogenesis of many disease states, the interaction between the two is poorly understood. Cyclopentenone isoprostanes (IsoPs), highly reactive structural isomers of the bioactive cyclopentenone prostaglandins PGA 2 and PGJ 2 , are formed non-enzymatically as products of oxidative stress in vivo. We have, for the first time, examined the effects of synthetic 15-A 2 -and 15-J 2 -IsoPs, two groups of endogenous cyclopentenone IsoPs, on the inflammatory response in RAW264.7 and primary murine macrophages. Cyclopentenone IsoPs potently inhibited lipopolysaccharide-stimulated IB␣ degradation and subsequent NF-B nuclear translocation and transcriptional activity. Expression of inducible nitric-oxide synthase and cyclooxygenase-2 were also inhibited by cyclopentenone IsoPs as was nitrite and prostaglandin production (IC 50 ϳ 360 and 210 nM, respectively). 15-J 2 -IsoPs potently activated peroxisome proliferator-activated receptor ␥ (PPAR␥) nuclear receptors, whereas 15-A 2 -IsoP did not, although the anti-inflammatory effects of both molecules were PPAR␥-independent. Interestingly 15-A 2 -IsoPs induced oxidative stress in RAW cells that was blocked by the antioxidant 4-hydroxy-TEMPO (TEMPOL) or the mitochondrial uncoupler carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone. TEMPOL also abrogated the inhibitory effect of 15-A 2 -IsoPs on lipopolysaccharide-induced NF-B activation, inducible nitricoxide synthase expression, and nitrite production, suggesting that 15-A 2 -IsoPs inhibit the NF-B pathway at least partially via a redoxdependent mechanism. 15-J 2 -IsoP, but not 15-A 2 -IsoP, also potently induced RAW cell apoptosis again via a PPAR␥-independent mechanism. These findings suggest that cyclopentenone IsoPs may serve as negative feedback regulators of inflammation and have important implications for defining the role of oxidative stress in the inflammatory response.

Pneumolysin (PLY) is a major virulence factor released by Streptococcus pneumoniae and has been implicated in the pathogenesis of pneumococcal pneumonia. In this study, we evaluated the contribution of newly recruited neutrophils and monocytes and resident alveolar macrophages to the pathogenesis of PLY-induced lung injury. Mice received either adhesion-blocking Abs to inhibit alveolar leukocyte trafficking or liposomal clodronate to deplete alveolar macrophages before intratracheal application of native PLY or its noncytotoxic derivative PdB. We found that treatment with PLY but not PdB resulted in increased lung vascular permeability. In addition, PLY also induced a decrease in the resident alveolar macrophage population, and the recruitment of peripheral blood neutrophils and monocytes into the alveolar space. Blockade of PLY-induced alveolar leukocyte trafficking by pretreatment of mice with anti-CD18 plus anti-CD49d Abs or depletion of circulating neutrophils did not attenuate the increase in lung permeability observed in response to intratracheal PLY. In addition, depletion of resident alveolar macrophages with clodronated liposomes did not reduce alveolar injury developing in response to PLY. PLY-induced lung injury was associated with only a small increase in bronchoalveolar lavage concentrations of cytokines. These data indicate that PLY-induced lung injury results from direct pneumotoxic effects on the alveolar-capillary barrier and is independent of both resident and recruited phagocytic cells.

The CC chemokine ligand-2 (CCL2) and its receptor CCR2 are essential for monocyte trafficking under inflammatory conditions. However, the mechanisms that determine the intensity and duration of alveolar monocyte accumulation in response to CCL2 gradients in inflamed lungs have not been resolved. To determine the potential role of CCR2-expressing monocytes in regulating alveolar CCL2 levels, we compared leukocyte recruitment kinetics and alveolar CCL2 levels in wild-type and CCR2-deficient mice in response to intratracheal LPS challenge. In wild-type mice, LPS elicited a dose- and time-dependent alveolar monocyte accumulation accompanied by low CCL2 levels in bronchoalveolar lavage fluid (BALF). In contrast, LPS-treated CCR2-deficient mice lacked alveolar monocyte accumulation, which was accompanied by relatively high CCL2 levels in BALF. Similarly, wild-type mice that were treated systemically with the blocking anti-CCR2 antibody MC21 completely lacked LPS-induced alveolar monocyte trafficking that was associated with high CCL2 levels in BALF. Intratracheal application of anti-CCR2 antibody MC21 to locally block CCR2 on both resident and recruited cells did not affect LPS-induced alveolar monocyte trafficking but led to significantly increased BALF CCL2 levels. Reciprocally bone marrow-transplanted, LPS-treated wild-type and CCR2-deficient mice showed a strict inverse relationship between alveolar monocyte recruitment and BALF CCL2 levels. In addition, freshly isolated human and mouse monocytes were capable of integrating CCL2 in vitro. LPS-induced alveolar monocyte accumulation is accompanied by monocytic CCR2-dependent consumption of CCL2 levels in the lung. This feedback loop may limit the intensity of monocyte recruitment to inflamed lungs and play a role in the maintenance of homeostasis.

S U M M A R YWe investigated the effects of Gram-negative bacterial lipopolysaccharide (LPS) on luciferase expression in transgenic reporter mice in which luciferase expression is driven by the nuclear factor B (NF-B)-dependent portion of the human immunodeficiency virus-1 (HIV-1) long terminal repeat (HIV-1 LTR). Using these mice, we dissected the sources of luciferase activity at the organ level by (a) assessing luciferase activity in organ homogenates, (b) bioluminescence imaging in vivo, and (c) bioluminescence imaging of individual organs ex vivo. Luciferin dosage was a critical determinant of the magnitude of photon emission from these reporter mice. Photon emission increased at doses from 0.5-6 mg of luciferin given by intraperitoneal (IP) injection. The differential between basal and LPS-induced bioluminescence was maximal at 3-6 mg of luciferin. Luciferase expression was highly inducible in lungs, liver, spleen, and kidneys after a single IP injection of LPS, as assessed by luciferase activity measurements in organ homogenates. Luciferase activity was also induced in the forebrain by treatment with IP LPS. In contrast, aerosolized LPS produced a response localized to the lungs as assessed by both bioluminescence and ex vivo luciferase assay measurements. These studies demonstrate the utility of luciferase reporter mice for determining organ-specific gene expression in response to local and systemic stimuli.