Michael W. Owens

Nitric oxide synthase in circulating vs. extravasated polymorphonuclear leukocytes

It is becoming increasingly apparent that certain forms of acute and chronic inflammation are associated with enhanced production of nitric oxide (NO). Although substantial information has been obtained describing the regulation of NO synthase (NOS) in macrophages, little information is available regarding the biochemistry and molecular biology of NOS in circulating vs. extravasated polymorphonuclear leukocytes (PMNs). The objective of this study was to characterize the molecular and biochemical properties of the inducible NO synthase (iNOS) in circulating vs. extravasated rat and human PMNs. Circulating rat and human PMNs were purified from peripheral blood and extravasated PMNs were elicited in rats by intraperitoneal injection of 1% oyster glycogen or in humans by peritoneal dialysis of patients with peritonitis. Inducible NOS mRNA from circulating and elicited PMNs was quantified using slot blot hybridization analysis with a cDNA probe specific for iNOS. iNOS protein was identified using Western immunoblot analysis, and NOS activity was quantified by measuring the NG‐monomethyl‐L‐arginine (L‐NMMA)‐inhibitable conversion of 14C‐labeled L‐arginine to L‐[14C]citrulline. In a separate series of experiments, circulating or extravasated PMNs were cultured for 4 h and the accumulation of L‐NMMA‐inhibitable nitrite (NO2−) in the supernatant was determined and used as a measure of NO production in vitro. We found that circulating PMNs (rat or human) contained no iNOS mRNA, protein, or enzymatic activity. Furthermore, circulating rat or human PMNs (2 × 106 cells/well) were unable to generate significant amounts of NO2− when cultured for 4 h in vitro. In contrast, iNOS mRNA levels in 4‐ and 6‐h elicited rat PMNs increased 21‐ and 42‐fold, respectively, when compared with circulating cells. Western blot analysis revealed the presence of iNOS protein in the elicited rat PMNs and iNOS enzymatic activity increased from normally undetectable levels in circulating rat PMNs to 81 and 285 pmol/min/mg for the 4‐ and 6‐h elicited rat PMNs, respectively. Approximately 20–30% of the total iNOS activity was Ca2+‐dependent. Nitrite formation by elicited rat PMNs in the absence of any exogenous stimuli increased from normally undetectable amounts for circulating PMNs to approximately 8 and 11 μM/106 cells for the 4‐ and 6‐h elicited PMNs, respectively. Highly enriched preparations of extravasated human PMNs contained neither message, protein nor iNOS enzymatic activity. Taken together our data demonstrate that inflammation‐induced extravasation of rat PMNs upregulates the transcription and translation of iNOS in a time‐dependent fashion and that 20–30% of the total inducible NOS is Ca2+‐dependent. In contrast, neither circulating nor extravasated human PMNs contained iNOS message, protein, or enzymatic activity. These data suggest that the human PMN iNOS gene is under very different regulation than is the rat gene.

Elevation of nitrotyrosine and nitrate concentrations in cystic fibrosis sputum

Nitric oxide (NO) is increased in the exhaled air of some patients with inflammatory lung disorders, but not in others. NO may combine with superoxide to form peroxynitrite, which lowers NO gas concentrations, increases formation of nitrate, and increases nitration of tyrosine residues on proteins. We hypothesized that superoxide released from neutrophils in the lower respiratory tract of cystic fibrosis (CF) results in increased nitrate and nitrotyrosine levels in sputum. In order to test this hypothesis, exhaled NO was collected from 5 stable adult CF subjects and from 5 nonsmoking normal controls.

Superoxide released from neutrophils causes a reduction in nitric oxide gas

Exhaled nitric oxide (NO) is increased in some inflammatory airway disorders but not in others such as cystic fibrosis and acute respiratory distress syndrome. NO can combine with superoxide ([Formula: see text]) to form peroxynitrite, which can decompose into nitrate. Activated polymorphonuclear neutrophils (PMNs) releasing[Formula: see text] could account for a reduction in exhaled NO in disorders such as cystic fibrosis. To test this hypothesis in vitro, we stimulated confluent cultures of LA-4 cells, a murine lung epithelial cell line, to produce NO. Subsequently, human PMNs stimulated to produce [Formula: see text] were added to the LA-4 cells. A gradual increase in NO in the headspace above the cultures was observed and was markedly reduced by the addition of PMNs. An increase in nitrate in the culture supernatant fluids was measured, but no increase in nitrite was detected. Superoxide dismutase attenuated the PMN effect, and xanthine/xanthine oxidase reproduced the effect. No changes in epithelial cell inducible NO synthase protein or mRNA were observed. These data demonstrate that [Formula: see text]released from PMNs can decrease NO by conversion to nitrate and suggest a potential mechanism for modulation of NO levels in vivo.

Cytokines increase proliferation of human intestinal smooth muscle cells: possible role in inflammation-induced stricture formation.

Crohns disease is an idiopathic, chronic inflammation of the gastrointestinal tract that causes narrowing and stricturing of primarily the small and large intestine. Although the mechanism(s) by which chronic inflammation promotes stricture formation remain to be defined, it does appear to be associated histologically with a hyperplasia of smooth muscle cells and an increased deposition of collagen within the bowel wall. The objective of this study was to assess the effect of two proinflammatory cytokines, tumor necrosis factor and interleukin-1, on the proliferation of human intestinal smooth muscle cells in vitro. Human intestinal smooth muscle cells were seeded at subconfluent densities into 24-well plates in Dulbeccos modified Eagles medium supplemented with 10% fetal bovine serum. Human recombinant tumor necrosis factor (0.1–100 ng/ml), interleukin-1 (0.1–500 ng/ml), or control medium (without cytokines) was then added to the cells and incubation continued for 48 or 72 h. Proliferation was determined by the incorporation of tritiated thymidine, added during the final 18 h, into the cellular DNA of the smooth muscle cells. Both cytokines caused a significant dose-dependent increase in intestinal smooth muscle cell proliferation relative to control. These results suggest that the interleukin-1 and tumor necrosis factor produced during chronic inflammation in vivo may enhance the proliferation of smooth muscle cells within the intestinal bowel wall and hence potentially contribute to the narrowing and stricturing of the intestine that is observed in Crohns disease.

Inhibition of pleural mesothelial cell collagen synthesis by nitric oxide

The pleural mesothelial cell has a critical role in repairing the mesothelium after injury via its ability to produce connective tissue macromolecules. We have recently shown that proinflammatory cytokines and lipopolysaccharide induce pleural mesothelial cells to produce nitric oxide. The present study examined the effect of nitric oxide on pleural mesothelial cell protein synthesis. Rat pleural mesothelial cells were exposed to various combinations of tumor necrosis factor, interleukin-1, interferon-gamma, and lipopolysaccharide or to the nitric oxide donors: 6-morpholino-sydnonimine, S-nitroso-N-acetyl-D,L-penicillamine, sodium nitroprusside, and spermine-NO adduct for 24-48 h. Nitrate and nitrite (an index of nitric oxide production) and not collagen and noncollagen protein production (uptake of 3H-proline into collagenase-sensitive protein) were then determined. Net collagen production was significantly inhibited by the cytokine-lipopolysaccharide combinations tested. Collagen inhibition paralleled the time course of increased nitric oxide production. The inhibition of collagen production was also significantly reversed by the addition of NG-nitro-L-arginine methyl ester, and was reproduced by the addition of a 5:1 molar excess of L-arginine to NG-nitro-L-arginine methyl ester. Additionally, nitric oxide-generating compounds significantly inhibited collagen production in a dose-dependent manner compared to unexposed control cells. Net collagen production was inhibited to a greater degree than noncollagen protein synthesis. These results suggest that nitric oxide may be a significant mediator of PMC collagen production during conditions of significant pleural inflammation.

Growth factor modulation of rat pleural mesothelial cell mitogenesis and collagen synthesis

This study examined the effects of an epithelial and a mesenchymal growth factor on pleural mesothelial cell proliferation and collagen synthesis, functions that may be important in the response of the pleura to injury. Epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) added singly caused significant increases relative to control in both the uptake of [3H]thymidine into the cellular DNA of subconfluent monolayers and of [3H]proline into collagenase-sensitive protein. Combinations of EGF and PDGF resulted in more than additive increases in proliferation and additive increases in collagen production relative to each factor alone. Media from control and growth factor-stimulated PMC demonstrated no gelatinase or collagenase activity, suggesting that the increase in net collagen production was secondary to enhanced synthesis. These data demonstrate that both epithelial and mesenchymal growth factors can stimulate PMC proliferation and collagen synthesis and that these growth factors have even greater effects when combined, particularly in regard to cellular proliferation. Increases in PMC proliferation and collagen synthesis in response to these growth factors may be important in healing the pleura after injury by a variety of disease processes.

MODULATION OF ENDOTHELIAL CELL PERMEABILITY BY LUNG CARCINOMA CELLS: A Potential Mechanism of Malignant Pleural Effusion Formation

This study examined the hypothesis that tumor cells metastatic to the pleura secrete a soluble factor(s) that directly increases endothelial cell permeability. Nitrocellulose filters were endothelialized with bovine pulmonary artery endothelial cells and exposed to conditioned media from either human lung adenocarcinoma (Calu-3), human lung squamous cell carcinoma (SK-MES-1), or control media for 16 h. The diffusional permeability (Pd × 10−5 cm/sec) to [14C]albumin was then determined for each monolayer with Ussing-type chambers. Both adenocarcinoma conditioned media (ACCM) and squamous cell carcinoma conditioned media (SCCM) caused a two- to threefold increase in endothelial monolayer permeability. The addition of indomethacin (10 μg/ml) blocked the observed permeability increase in ACCM but not in SCCM, suggesting that the increase in permeability by ACCM was secondary to the production of prostaglandins. To confirm this, a variety of prostanoids previously shown to be produced by the Calu-3 cell line were added directly to the endothelial monolayer. Prostaglandin F2α (PGF2α) in both low (10 ng/ml) and high (100 ng/ml) concentrations for 16 h resulted in a three- to fourfold increase in permeability. Prostaglandin E2 (PGE2) resulted in a small increase in [14C]albumin permeability but only at high concentrations (100 ng/ml). PGF2α production by the two tumor cell lines was measured using radioimmunoassay. Baseline adenocarcinoma production of PGF2α was 117.5 pmol/106 cells and fell to 24.2 pmol/106 cells hours following incubation with indomethacin. The decrease in PGF2α occurred in parallel with the changes in permeability. Concomitant, reversible changes in cell shape and F-actin distribution were detected in endothelial cells exposed to ACCM. No significant production of PGF2α by the squamous cell carcinoma cell line was detected. These results suggest that both adenocarcinoma and squamous cell carcinoma secrete a soluble factor(s) that directly increases endothelial cell permeability to albumin and that in the case of adenocarcinoma this soluble factor may be a prostanoid such as PGF2α.This study examined the hypothesis that tumor cells metastatic to the pleura secrete a soluble factor(s) that directly increases endothelial cell permeability. Nitrocellulose filters were endothelialized with bovine pulmonary artery endothelial cells and exposed to conditioned media from either human lung adenocarcinoma (Calu-3), human lung squamous cell carcinoma (SK-MES-1), or control media for 16 h. The diffusional permeability (Pd × 10−5 cm/sec) to [14C]albumin was then determined for each monolayer with Ussing-type chambers. Both adenocarcinoma conditioned media (ACCM) and squamous cell carcinoma conditioned media (SCCM) caused a two- to threefold increase in endothelial monolayer permeability. The addition of indomethacin (10 μg/ml) blocked the observed permeability increase in ACCM but not in SCCM, suggesting that the increase in permeability by ACCM was secondary to the production of prostaglandins. To confirm this, a variety of prostanoids previously shown to be produced by the Calu-3 cell line were added directly to the endothelial monolayer. Prostaglandin F2α (PGF2α) in both low (10 ng/ml) and high (100 ng/ml) concentrations for 16 h resulted in a three- to fourfold increase in permeability. Prostaglandin E2 (PGE2) resulted in a small increase in [14C]albumin permeability but only at high concentrations (100 ng/ml). PGF2α production by the two tumor cell lines was measured using radioimmunoassay. Baseline adenocarcinoma production of PGF2α was 117.5 pmol/106 cells and fell to 24.2 pmol/106 cells hours following incubation with indomethacin. The decrease in PGF2α occurred in parallel with the changes in permeability. Concomitant, reversible changes in cell shape and F-actin distribution were detected in endothelial cells exposed to ACCM. No significant production of PGF2α by the squamous cell carcinoma cell line was detected. These results suggest that both adenocarcinoma and squamous cell carcinoma secrete a soluble factor(s) that directly increases endothelial cell permeability to albumin and that in the case of adenocarcinoma this soluble factor may be a prostanoid such as PGF2α.

Sandblaster's lung with mycobacterial infection

This report describes the development of alveolar silico-lipoproteinosis complicated by Mycobacterium kansasii infection in a previously healthy man who worked as a sandblaster. Alveolar silico-lipoproteinosis is a rare disease that usually is fatal within 1 year of onset of symptoms. There is a high incidence of mycobacterial infection, half being caused by atypical organisms.

A Home Telemonitoring Program Reduced Exacerbation and Healthcare Utilization Rates in COPD Patients with Frequent Exacerbations

RATIONALE As the impact of home telemonitoring on patients with chronic obstructive pulmonary disease (COPD) is not fully understood and reduction in healthcare utilization is not proven, we attempt to evaluate the effects of home telemonitoring on healthcare utilization in patients with COPD. SUBJECTS AND METHODS We conducted a retrospective cohort study using the Veterans Health Administration database of COPD patients enrolled in the Care Coordination Home Telehealth (CCHT) program. We evaluated the effects of monitoring through this program in patients with moderate to severe COPD and frequent exacerbations. Numbers of emergency department (ED) visits, urgent care (UC) visits, and hospitalizations were all evaluated before and after enrollment. The differences in average pre-enrollment and during-enrollment numbers of hospital admissions, ED/UC visits, and exacerbations were tested for significance among all patients enrolled in the program who had one or more exacerbations at pre-enrollment; results were expressed on a per-year basis. RESULTS Data were available on a total of 1,133 patients with COPD enrolled in the CCHT program between 2005 and 2009. Given the objectives of our study, we only included 369 patients who had at least one exacerbation per year in the year prior to enrollment. Of these, 71.5% had a reduction in numbers of ED visits and exacerbations requiring hospitalizations after enrollment in the program. The average number of hospital admissions, ED visits, and total exacerbations were all reduced (0.41 ± 1.68, 0.15 ± 1.65, and 0.56 ± 2.3, respectively; all with p<0.01). The pre-enrollment number of exacerbations was the only factor observed to be significantly associated with the reduction in number of exacerbations. CONCLUSIONS In patients with COPD and frequent exacerbations, enrollment in a home telemonitoring program may decrease healthcare utilization.

Nitric Oxide Synthesis by Rat Pleural Mesothelial Cells: Induction by Growth Factors and Lipopolysaccharide

The purpose of this study was to determine if certain growth factors and bacterial products induce pleural mesothelial cells (PMC) to produce nitric oxide (NO). Confluent monolayers of rat PMC were exposed to epidermal growth factor (EGF), platelet-derived growth factor (PDGF), or lipopolysaccharide (LPS) individually and in various combinations for 24-72 h. Concentrations of nitrite and nitrate were quantified and used as an indirect measure of NO production. LPS stimulation resulted in a significant increase in nitrite/nitrate concentration, but neither EGF nor PDGF alone or combined had any significant effect relative to control. However, LPS combined with either EGF or PDGF caused a significant increase in nitrite/nitrate concentration relative to LPS alone and growth factor alone. The highest level level of nitrite/nitrate concentration was observed with the triple combination of LPS, EGF, and PDGF. Nitrite/nitrate accumulation was significantly increased at 24 h by all combinations, and continued to increase, with the highest concentration observed after 72 h of exposure. Nitrite/nitrate production was significantly inhibited by NG-nitro-L-arginine methyl ester and this inhibition was reversed by the addition of L-arginine, suggesting that nitrite and nitrate were derived from the L-arginine-dependent formation of NO. These data indicate that PMC can be induced to produce relatively large amounts of NO in response to growth factors combined with LPS.