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We focused on the regulation of inflammatory mediator expression by adenovirus E1A in lung epithelial cells and the role of this viral protein in the pathogenesis of chronic obstructive pulmonary disease (COPD). We previously reported that E1A, a well-known regulator of host genes, increased ICAM-1 expression in human bronchial epithelial (HBE) and A549 cells in response to LPS stimulation. In this report, we clarified the mechanism of this regulation. We found NF-[kappa]B translocation to the nucleus after LPS stimulation in both E1A-positive and -negative HBE cells. ICAM-1 promoter reporter constructs revealed that a mutation in the proximal NF-[kappa]B binding site completely inhibited increased transcription, whereas the mutation in a distal site did not. We analyzed the participation of E1A in transcriptional complex formation at this promoter using chromatin immunoprecipitation. In E1A-positive HBE and A549 cells, LPS stimulation increased ICAM-1 promoter immunoprecipitation by NF-[kappa]B p65 and p300 but not activator protein-1 antibodies with a concomitant increase by the E1A antibody. No increase was found in E1A-negative cells except in HBE cells with p65 antibody. The association of E1A with the increased promoter immunoprecipitation with p300 was also observed after TNF-[alpha] stimulation of A549 cells. These results suggest that adenovirus E1A regulates the ICAM-1 promoter through its proximal NF-[kappa]B binding site, most likely by interacting with the transcriptional complex that forms at this site. E1A regulation of the LPS response may play a role in acute exacerbations as a consequence of bacterial infections in COPD. gene regulation; inflammation; nuclear factor-[kappa]B

Epidemiologic studies have shown an association between exposure to ambient particulate air pollution atherosclerosis; adhesion molecules

Inflammation accelerates polymorphonuclear leukocyte (PMN) release from the bone marrow, and these PMNs are implicated in inappropriate tissue injury. We have previously developed a method using 5'-bromo-2'deoxyuridine (BrdU) to study PMN kinetics using an immunocytochemical grading system of PMN on cytospin slides. The aim of this study was to develop a flow cytometric method to quantify the number of positively stained PMN and grade the intensity of staining for the transit time calculation of PMN through the marrow. Dividing myeloid progenitors in the marrow of rabbits were labeled with a pulse dosage of intravenous BrdU. BrdU-labeled PMN ([PMN.sup.BrdU]) were detected in the circulation using a FITC-conjugated anti-BrdU monoclonal antibody. The [PMN.sup.BrdU] were assigned to five groups according to their FITC intensity, and the transit times of PMN at different stages of development in the marrow were calculated. Results were compared using parallel immunocytochemical analysis of the same samples. In control animals, [PMN.sup.BrdU] in the circulation peaked at 72 h after BrdU labeling with 36.0% of PMN labeled. In normal rabbits, the transit times of PMN through the mitotic pool (49.5 [+ or -] 4.2 h) and maturation pool (65.5 [+ or -] 3.1 h) correlated well with immunocytochemical analysis and previously published values. Using this method, we demonstrated that exposure to air pollution particles accelerates the release of [PMN.sup.BrdU] from the marrow. We conclude that a flow cytometric approach for identifying BrdU-labeled leukocytes provides an objective and accurate method for studying leukocyte kinetics and behavior. polymolphonuclear neutrophil; flow cytometry; transit time

Alveolar macrophages (AM) play a key role in clearing atmospheric particulates from the lung surface and stimulating epithelial cells to produce proinflammatory mediators. The present study examines the role of 'acute response' cytokines TNF-[alpha] and IL-1[beta] released by AM exposed to ambient particulate matter with a diameter of 0.05), and the combination of the two antibodies most effective. When HBEC were treated similarly, anti-TNF-[alpha] antibodies had the greatest effect. In A549 cells P[M.sub.10]-exposed AM supernatants increased NF-[kappa]B, activator protein (AP)-1 and specificity protein 1 binding, while anti-TNF-[alpha] and anti-IL-1[beta] antibodies reduced NF-[kappa]B and AP-1 binding. We conclude that AM-derived TNF-[alpha] and IL-1[beta] provide a major stimulus for the production of proinflammatory mediators by lung epithelial cells and that their relative importance may depend on the type of epithelial cell target. ribonuclease protection assay; transcription factors; enzyme-linked immunosorbent assay; proinflammatory mediators; particulate matter with diameter < 10 [micro]m

Exposure to air pollution [particulate matter, particles air pollution; alveolar macrophages; atherosclerosis; inflammation; leukocytes

Previous studies showed an association between latent adenoviral infection with expression of the adenoviral E1A gene and chronic obstructive pulmonary disease (COPD). The present study focuses on how the adenoviral EIA gene could alter expression of growth factors by human bronchial epithelial (HBE) cells. The data show that connective tissue growth factor (CTGF) and transforming growth factor (TGF)-[beta]1 mRNA and protein expression were upregulated in E1A-positive HBE cells. Upregulation of CTGF in this in vitro model was independent of TGF-[beta] secreted into the growth medium. Comparison of E1A-positive with EIA-negative HBE cells showed that both expressed cytokeratin but only EIA-positive cells expressed the mesenchymal markers vimentin and [alpha]-smooth muscle actin. We conclude that latent infection of epithelial cells by adenovirus E1A could contribute to airway remodeling in COPD by the viral E1A gene, inducing TGF-[beta]1 and CTGF expression and shifting cells to a more mesenchymal phenotype. bronchial epithelial cell; differentiation; transforming growth factor-[beta]; connective tissue growth factor; chronic obstructive pulmonary disease

The present study was designed to develop methods to study the production and release of monocytes from the bone marrow using the thymidine analog 5'-bromo-2'-deoxyuridine (BrdU). Dividing monocytes in bone marrow were labeled with BrdU ([MO.sup.BrdU]), and their release into the blood and disappearance from the circulation were monitored using a double immunostaining method. The first [MO.sup.BrdU] appeared in the circulation 4 h after labeling with BrdU and peaked at 18 h when 34.3 [+ or -] 5.8% of monocytes were labeled. The calculated transit time of monocytes through bone marrow was 38.1 [+ or -] 3.1 h in control rabbits with a half-life ([T.sub.1/2]) of 12.7 h. Instillation of Streptococcus pneumoniae into the lung accelerated the release of monocytes from bone marrow (peak at 10 h) and shortened their bone marrow transit time (27.1 [+ or -] 1.8 vs. 22.6 [+ or -] 0.6, vehicle vs. pneumonia; P < 0.05). We conclude that this nonradioisotope method provides a novel way to monitor monocyte kinetics and confirmed previous reports that a focal pneumonia shortens monocyte marrow transit and increases their release into the circulation. leukocytes; transit time; half life; pneumonia; rabbits

We examined the hypothesis that ambient particulate matter with a diameter of interleukin-8; monocyte chemoattractant protein-1; intercellular adhesion molecule-1; nuclear factor-[kappa]B; transcription factor specificity protein 1; particulate matter

Interleukin-6 (IL-6) shortens the transit time of polymorphonuclear leukocytes (PMN) through the marrow and accelerates their release into the circulation. In contrast to other inflammatory stimuli, this response is associated with a decrease in L-selectin levels on circulating PMN. The present study was designed to determine the effect of IL-6 on L-selectin levels of PMN in rabbits. Recombinant human IL-6 (2 [micro]g/kg) caused a decrease in L-selectin levels on circulating PMN 3 to 12 h after treatment (P < 0.05). L-selectin levels decreased on PMN already in the circulation for up to 4 h (P < 0.05), on PMN released from the marrow posttreatment for up to 12 h (P < 0.01) and on PMN in the marrow for up to 6 h (P < 0.05) after IL-6 treatment. We conclude that IL-6 decreases L-selectin levels on circulating PMN by demarginating PMN with low levels of L-selectin and by releasing PMN from the marrow with low levels of L-selectin. We postulate that this prolonged downregulation of L-selectin on circulating PMN could influence their recruitment into inflammatory sites. cytokine; bone marrow; neutrophil

Experiments were performed to investigate the migration of polymorphonuclear leukocytes (PMN) or neutrophils from the bone marrow into the lung tissue succeeding bacteremic infection. The bone marrow release and sequestration of PMNs during a focal subcutaneous infection were measured using immunohistochemistry. Granulocytopenia was observed following the infection and was attributed to the release of younger and immature PMNs in the circulation.

A study was conducted on cytoskeleton function and the persistent white blood cell deformation in pulmonary capillaries. Experiments on isolated rabbit lungs show that white blood cells are retained preferentially over erythrocytes. Results also show that leukocyte deformation continued even after cells left pulmonary circulation. It was concluded that there are very few changes in the cytoskeleton during white blood cell deformation.

The expression of L-selectin on polymorphonuclear lymphocytes (PMNs) from the hemopoietic and sinusoidal compartments of the bone marrow was analyzed to determine the role of L-selectin in PMN release. Analysis of bone marrow and peripheral blood samples from patients after normothermic cardiopulmonary bypass indicated the presence of elevated L-selectin expression in the sinusoidal compartments. Furthermore, the presence of L-selectin in the sinusoids regulated the release of PMNs from the bone marrow.

Mature polymorphonuclear leukocytes (PMNs) isolated from New Zealand White rabbits were analyzed by flow cytometry to determine effects of senescence on L-selectin expression in PMNs. Analysis of rabbit PMNs by flow cytometry indicated the presence of decreasing levels of L-selectin expression due to cellular senescence. Older circulating PMNs also exhibited constitutive shedding of L-selectin due to interactions with the endothelium in the microvessels.

The transit times of polymorphonuclear leukocytes (PMN) in the mitotic and postmitotic pools of rabbit bone marrow (BM) were analyzed by utilizing 5'-bromo-2'-deoxyuridine thymidine analogues. The total PMN transit time in rabbit BM is 95 hours. PMN transit time in the mitotic pools of the BM is 51 hours while the PMN transit time in the postmitotic or maturation pool is 65 hours. However, the local pneumonic process decreases the transit time in the mitotic and maturation pools indicating the role of PMN in the pathogenesis of lung injury.

Bacterial pathogens produce endotoxins that damage the heart by disrupting the contractile ability of cardiomuscular tissue. To determine whether the reduction in contractility is due to factors circulated in the blood, an isolated rabbit heart was supplied through a Langendorff column with blood from a rabbit treated with endotoxin. The isolated heart showed a abnormalities in muscle cells, interstitial edema and filling of the capillaries with leukocytes after five hours of endotoxin treatment.

Administration of endotoxin in a pig model of hyperdynamic sepsis reduces myocardial leukocyte transit by 5-40 s thereby retaining 2 x 10 to the power 9 leukocytes in coronary microcirculation. The leukocytes are retained in the capillaries and not in the postcapillary venules. The number and position of retained myocardial leukocytes is determined by postmortem morphometric analysis.

Tissue macrophages and interstitial airways generate platelet-derived growth factor (PDGF), which activates human eosinophils to produce inflammation of the airways in asthma and chronic obstructive pulmonary diseases (COPD). PDGF multiplies smooth muscle cells and is mitogenic for cultured fibroblasts. Asthmatic patients reveal higher levels of mRNA for PDGF and PDGF-beta than COPD patients due to the glucocorticoid administration.

The presence of the adenoviral early region 1A (E1A) protein in human lungs has been associated with an increased risk of chronic obstructive pulmonary disease (COPD), possibly by a mechanism involving amplification of proinflammatory responses. We hypothesize that enhanced inflammation results from increased transcription factor activation in E1A-carrying cells, which may afford susceptibility to environmental particulate matter [is less than] 10 [micro]m ([PM.sub.10])-mediated oxidative stress. We measured interleukin (IL)-8 mRNA expression and protein release in human alveolar epithelial cells (A549) transfected with the E1A gene (E1A+ve). Both E1A+ve and -ve cells released IL-8 after incubation with TNF-[Alpha], but only E1A+ve cells were sensitive to LPS stimulation in IL-8 mRNA expression and protein release. E1A+ve cells showed an enhanced IL-8 mRNA and protein response after treatment with [H.sub.2][O.sub.2] and [PM.sub.10]. E1A-enhanced induction of IL-8 was accompanied by increases in activator protein-1 and nuclear factor-[Kappa]B nuclear binding in E1A+ve cells, which also showed higher basal nuclear binding of these transcription factors. These data suggest that the presence of E1A primes the cell transcriptional machinery for oxidative stress signaling and therefore facilitates amplification of proinflammatory responses. By this mechanism, susceptibility to exacerbation of COPD in response to particulate air pollution may occur in individuals harboring E1A. early region 1A; environmental particulate matter less than 10 micrometers; interleukin-8; nuclear factor-[Kappa]B; activator protein-1; lung epithelium

Suwa, Tatsushi, James C. Hogg, Dean English, and Stephan F. van Eeden. Interleukin-6 induces demargination of intravascular neutrophils and shortens their transit in marrow. Am J Physiol Heart Circ Physiol 279: H2954-H2960, 2000.--Recombinant human interleukin-6 (IL-6) causes both a thrombocytosis and leukocytosis. The thrombocytosis is caused by an accelerating thrombocytopoiesis, but the mechanism of the leukocytosis is unknown. This study was designed to determine the relative contributions of marrow stimulation and intravascular demargination to the IL-6 induced neutrophilia. IL-6 (2 [micro]g/kg), administered intravenously to rabbits, caused a biphasic neutrophilia with an initial peak at 3 h and a second peak at 9 h. Using the thymidine analog 5'-bromo-2'-deoxyuridine (BrdU) to label dividing polymorphonuclear leukocytes (PMNs) in the bone marrow, we showed that IL-6 treatment mobilizes PMNs from the marginated pool into the circulating pool at 2-6 h with a decrease in L-selectin expression on PMNs and also accelerates the release of PMNs from the postmitotic pool in the bone marrow at 12-24 h. We have concluded that IL-6 causes a biphasic neutrophilia wherein the first peak results from the mobilization of PMNs into the circulating pool from the marginated pool and the second peak results from an accelerated bone marrow release of PMNs. neutrophilia; cytokine; bone marrow; L-selectin

Mukae, Hiroshi, James C. Hogg, Dean English, Renaud Vincent, and Stephan F. van Eeden. Phagocytosis of particulate air pollutants by human alveolar macrophages stimulates the bone marrow. Am J Physiol Lung Cell Mol Physiol 279: L924-L931, 2000.--Epidemiologic studies have shown an association between the level of ambient particulate matter [is less than] 10 [micro] m ([PM.sub.10]) and cardiopulmonary mortality. We have shown that exposure of rabbits to P[M.sub.10] stimulates the bone marrow. In this study, we determined whether human alveolar macrophages (AMs) that phagocytose atmospheric [PM.sub.10] produce mediators capable of stimulating the bone marrow. AMs incubated with [PM.sub.l0] for 24 h produced tumor necrosis factor-[Alpha] in a dose-dependent manner (86.8 [+ or -] 53.29 pg/ml with medium alone; 1,087.2 [+ or -] 257.3 pg/ml with 0.1 mg/ml of P[M.sub.10]; P [is less than] 0.02). Instillation of the supernatants from AMs incubated with 0.1 mg/ml of [PM.sub.10] into the lungs of rabbits (n = 6) increased circulating polymorphonuclear leukocyte (PMN) and band cell counts as well as shortened the PMN transit time through the bone marrow (87.9 [+ or -] 3.3 h) compared with unstimulated human AMs (104.9 [+ or -] 2.4 h; P [is less than] 0.01; n = 5 rabbits). The supernatants from rabbit AMs incubated with 0.1 mg/ml of [PM.sub.10] (n = 4 rabbits) caused a similar shortening in the PMN transit time through the bone marrow (91.5 [+ or -] 1.6 h) compared with human AMs. We conclude that mediators released from AMs after phagocytosis of [PM.sub.10] induce a systemic inflammatory response that includes stimulation of the bone marrow. particulate matter less than 10 micrometers; air pollution; bone marrow; leukocytes; cytokines

Isolated perfuse rabbit hearts were used in the investigation of the mechanism of leukocyte retention in the myocardial capillary endothelium after endotoxin infusion. Leukocyte activation by itself did not increase leukocyte transit time and retention. However, endothelial response to inflammation increased both leukocyte transit time and retention. This indicates that changes in the endothelial cells secondary to inflammation also affect leukocyte transit in the coronary epithelium.