Exosomes in immunoregulation of chronic lung diseases.

Exosomes are nano-sized, membrane-bound vesicles released from cells that transport cargo including DNA, RNA, and proteins, between cells as a form of intercellular communication. In addition to their role in intercellular communication, exosomes are beginning to be appreciated as agents of immunoregulation that can modulate antigen presentation, immune activation, suppression, and surveillance. This article summarizes how these multifaceted functions of exosomes may promote development and/or progression of chronic inflammatory lung diseases including asthma, chronic obstructive pulmonary disease, and pulmonary fibrosis. The potential of exosomes as a novel therapeutic is also discussed.

Oxidative protein cross-linking reactions involving L-tyrosine in transforming growth factor-beta1-stimulated fibroblasts.

The mechanisms by which ligand-stimulated generation of reactive oxygen species in nonphagocytic cells mediate biologic effects are largely unknown. The profibrotic cytokine, transforming growth factor-beta1 (TGF-beta1), generates extracellular hydrogen peroxide (H2O2) in contrast to intracellular reactive oxygen species production by certain mitogenic growth factors in human lung fibroblasts. To determine whether tyrosine residues in fibroblast-derived extracellular matrix (ECM) proteins may be targets of H2O2-mediated dityrosine-dependent cross-linking reactions in response to TGF-beta1, we utilized fluorophore-labeled tyramide, a structurally related phenolic compound that forms dimers with tyrosine, as a probe to detect such reactions under dynamic cell culture conditions. With this approach, a distinct pattern of fluorescent labeling that seems to target ECM proteins preferentially was observed in TGF-beta1-treated cells but not in control cells. This reaction required the presence of a heme peroxidase and was inhibited by catalase or diphenyliodonium (a flavoenzyme inhibitor), similar to the effect on TGF-beta1-induced dityrosine formation. Exogenous addition of H2O2 to control cells that do not release extracellular H2O2 produced a similar fluorescent labeling reaction. These results support the concept that, in the presence of heme peroxidases in vivo, TGF-beta1-induced H2O2 production by fibroblasts may mediate oxidative dityrosine-dependent cross-linking of ECM protein(s). This effect may be important in the pathogenesis of human fibrotic diseases characterized by overexpression/activation of TGF-beta1.

Reactive oxygen species in cell signaling.

Reactive oxygen species (ROS) are generated as by-products of cellular metabolism, primarily in the mitochondria. When cellular production of ROS overwhelms its antioxidant capacity, damage to cellular macromolecules such as lipids, protein, and DNA may ensue. Such a state of "oxidative stress" is thought to contribute to the pathogenesis of a number of human diseases including those of the lung. Recent studies have also implicated ROS that are generated by specialized plasma membrane oxidases in normal physiological signaling by growth factors and cytokines. In this review, we examine the evidence for ligand-induced generation of ROS, its cellular sources, and the signaling pathways that are activated. Emerging concepts on the mechanisms of signal transduction by ROS that involve alterations in cellular redox state and oxidative modifications of proteins are also discussed.

Ras-dependent and -independent regulation of reactive oxygen species by mitogenic growth factors and TGF-beta1.

Mitogenic growth factors and transforming growth factor beta1 (TGF-beta1) induce the generation of reactive oxygen species (ROS) in nonphagocytic cells, but their enzymatic source(s) and regulatory mechanisms are largely unknown. We previously reported on the ability of TGF-beta1 to activate a cell surface-associated NADH:flavin:O(2) oxidoreductase (NADH oxidase) that generates extracellular H(2)O(2). In this study, we compared the ROS-generating enzymatic systems activated by mitogenic growth factors and TGF-beta1 with respect to the primary reactive species produced (O(2)(.-) vs. H(2)O(2)), the site of generation (intracellular vs. extracellular) and regulation by Ras. We find that the mitogenic growth factors PDGF-BB, FGF-2, and TGF-alpha (an EGF receptor ligand) are able to rapidly (within 5 min) induce the generation of intracellular O(2)(.-) without detectable NADH oxidase activity or extracellular H(2)O(2) release. In contrast, TGF-beta1 does not stimulate intracellular O(2)(.-) production and the delayed induction of extracellular H(2)O(2) release is not associated with O(2)(.-) production. Expression of dominant-negative Ras (N17Ras) protein by herpes simplex virus-mediated gene transfer blocks mitogen-stimulated intracellular O(2)(.-) generation but has no effect on TGF-beta1-induced NADH oxidase activation/H(2)O(2) production. These results demonstrate that there are at least two distinctly different ROS-generating enzymatic systems in lung fibroblasts regulated by mitogenic growth factors and TGF-beta1 via Ras-dependent and -independent mechanisms, respectively. In addition, these findings suggest that endogenous production of ROS by growth factors/cytokines may have different biological effects depending on the primary reactive species generated and site of production.

Transforming growth factor-beta 1-induced activation of the ERK pathway/activator protein-1 in human lung fibroblasts requires the autocrine induction of basic fibroblast growth factor.

Transforming growth factor-beta (TGF-beta) is involved in multiple processes including cell growth and differentiation. In particular, TGF-beta has been implicated in the pathogenesis of fibrotic lung diseases. In this study, we examined regulation of the mitogen-activated protein kinase pathway by TGF-beta1 in primary human lung fibroblasts. TGF-beta1 treatment resulted in extracellular signal-regulated kinase (ERK) pathway activation in a delayed manner, with maximal activity at 16 h. ERK activation occurred concomitantly with the induction of activator protein-1 (AP-1) binding, a nuclear factor required for activation of multiple genes involved in fibrosis. AP-1 binding was dependent on ERK activation, since the MEK-1 (mitogen-activated protein kinase kinase) inhibitor PD98059 inhibited TGF-beta1-induced binding. Induction of the receptor tyrosine kinase-linked growth factor, basic fibroblast growth factor (bFGF) protein expression temporally paralleled the activation of ERK/AP-1. Induction of AP-1 by TGF-beta1-conditioned medium was observed at 2 h, similar to AP-1 induction in response to exogenous bFGF. Dependence of ERK/AP-1 activation on bFGF induction was demonstrated by inhibition of TGF-beta1-induced ERK/AP-1 activation when conditioned medium from TGF-beta1-treated cells was incubated with bFGF-neutralizing antibody. Together, these results demonstrate that TGF-beta1 regulates the autocrine induction of bFGF, resulting in activation of the ERK mitogen-activated protein kinase pathway and induction of AP-1 binding.

Upregulated expression of fibroblast growth factor (FGF) receptors by transforming growth factor-beta1 (TGF-beta1) mediates enhanced mitogenic responses to FGFs in cultured human lung fibroblasts.

Transforming growth factor-beta1 (TGF-beta1) is a multifunctional cytokine that induces mesenchymal cell proliferation in vivo while inhibiting growth of most cells directly via serine-threonine receptor(s) binding/activation in vitro. In this study, the ability of TGF-beta1 to regulate the receptor expression of classical mitogenic growth factors that bind receptor tyrosine kinases was examined. TGF-beta1 markedly increased the protein expression of the fibroblast growth factor (FGF) receptors FGFR-1 (Flg) and FGFR-2 (Bek) in a time- and dose-dependent manner in human lung fibroblasts. This resulted in a potentiation of the mitogenic response of multiple FGF ligands that bind to these receptors. TGF-beta1 had no effect on epidermal growth factor (EGF) or platelet-derived growth factor (PDGF)-beta receptor expression and the mitogenic responses mediated by specific ligands for these receptors were not increased. These results demonstrate a novel action of TGF-beta1 to selectively upregulate the expression of FGF receptor family members leading to enhanced mitogenesis by FGFs.

Tyrosine phosphorylation regulates H2O2 production in lung fibroblasts stimulated by transforming growth factor beta1.

Transforming growth factor beta1 (TGF-beta1) is a multifunctional, profibrotic cytokine involved in cellular growth and differentiation. We have previously described a cell surface-associated H2O2-generating NADH:flavin:O2 oxidoreductase (referred to as NADH oxidase) activity in human lung fibroblasts induced by TGF-beta1 (Thannickal, V. J., and Fanburg, B. L. (1995) J. Biol. Chem. 270, 30334-30338). In this study, the potential for regulation of this novel TGF-beta1-activated oxidase in fibroblasts by protein tyrosine phosphorylation was examined. Immunoblots using anti-phosphotyrosine antibody demonstrated a time-dependent but delayed phosphorylation of two proteins of 115 and 103 kDa in cells stimulated with TGF-beta1 (2 ng/ml). Similar to the effect on TGF-beta1-induced H2O2 production, phosphorylation of these proteins was blocked by the addition of actinomycin D. The protein-tyrosine kinase inhibitors genistein and herbimycin A inhibited TGF-beta1-induced protein tyrosine phosphorylation, NADH oxidase activation, and H2O2 production in a dose-dependent manner. Catalase, diphenyliodonium (an inhibitor of flavoenzymes), and suramin (an inhibitor of receptor activation, added 4 h after TGF-beta1) had no effect on the induction of protein tyrosine phosphorylation. Phosphorylation of the 115- and 103-kDa proteins preceded the generation of H2O2 production and returned to control levels when H2O2 was undetectable at 48 h after TGF-beta1 exposure. These results suggest that protein tyrosine phosphorylation by a nonreceptor protein-tyrosine kinase(s) regulates the activity of the TGF-beta1-responsive H2O2-generating NADH oxidase in human lung fibroblasts. Additionally, this study demonstrates that TGF-beta1, which binds to a serine-threonine kinase receptor, is able to induce protein tyrosine phosphorylation in a delayed manner via a signaling pathway that requires transcriptional activation.

Activation of an H2O2-generating NADH oxidase in human lung fibroblasts by transforming growth factor beta 1.

The cellular source(s) and mechanisms of generation of reactive oxygen species (ROS) in nonphagocytic cells stimulated by cytokines are unclear. In this study, we demonstrate that transforming growth factor beta 1 (TGF-beta 1, 1 ng/ml) induces the release of H2O2 from human lung fibroblasts within 8 h following exposure to this cytokine. Elevation in H2O2 release peaked at 16 h (approximately 22 pmol/min/10(6) cells) and gradually declined to undetectable levels at 48 h after TGF-beta 1 treatment. NADH consumption by these cells was stimulated by TGF-beta 1 while that of NADPH remained unchanged. NADPH oxidase activity as measured by diphenyliodonium (DPI)-inhibitable NADH consumption in TGF-beta 1-treated cells followed a time course similar to that of H2O2 release. DPI, an inhibitor of the NADPH oxidase complex of neutrophils and other flavoproteins, also inhibited the TGF-beta 1-induced H2O2 production. Inhibitors of other enzymatic systems involving flavoproteins that may be responsible for the production of H2O2 in these cells, including xanthine oxidase, nitric oxide synthase, and both mitochondrial and microsomal electron transport systems, failed to inhibit TGF-beta 1-induced NADH oxidation and H2O2 production. The delay (> 4 h) following TGF-beta 1 exposure along with the inhibition of this process by cycloheximide and actinomycin D suggest the requirement of new protein synthesis for induction of NADH oxidase activity in TGF-beta 1-stimulated fibroblasts.

Nitric oxide decreases cytokine-induced endothelial activation. Nitric oxide selectively reduces endothelial expression of adhesion molecules and proinflammatory cytokines.

To test the hypothesis that nitric oxide (NO) limits endothelial activation, we treated cytokine-stimulated human saphenous vein endothelial cells with several NO donors and assessed their effects on the inducible expression of vascular cell adhesion molecule-1 (VCAM-1). In a concentration-dependent manner, NO inhibited interleukin (IL)-1 alpha-stimulated VCAM-1 expression by 35-55% as determined by cell surface enzyme immunoassays and flow cytometry. This inhibition was paralleled by reduced monocyte adhesion to endothelial monolayers in nonstatic assays, was unaffected by cGMP analogues, and was quantitatively similar after stimulation by either IL-1 alpha, IL-1 beta, IL-4, tumor necrosis factor (TNF alpha), or bacterial lipopolysaccharide. NO also decreased the endothelial expression of other leukocyte adhesion molecules (E-selectin and to a lesser extent, intercellular adhesion molecule-1) and secretable cytokines (IL-6 and IL-8). Inhibition of endogenous NO production by L-N-monomethyl-arginine also induced the expression of VCAM-1, but did not augment cytokine-induced VCAM-1 expression. Nuclear run-on assays, transfection studies using various VCAM-1 promoter reporter gene constructs, and electrophoretic mobility shift assays indicated that NO represses VCAM-1 gene transcription, in part, by inhibiting NF-kappa B. We propose that NO's ability to limit endothelial activation and inhibit monocyte adhesion may contribute to some of its antiatherogenic and antiinflammatory properties within the vessel wall.

Release of hydrogen peroxide in response to hypoxia-reoxygenation: role of an NAD(P)H oxidase-like enzyme in endothelial cell plasma membrane.

The dynamics and mechanisms of extracellular release of hydrogen peroxide (H2O2) from bovine pulmonary artery endothelial cells (EC) subjected to anoxia, hypoxia, and hypoxia followed by reoxygenation were examined using various inhibitors of enzymatic systems in intact cells and by direct measurement of H2O2 production from isolated EC plasma membranes. Extracellular H2O2 was measured with a fluorometric assay. EC exposed to hypoxia (3% O2) and anoxia (0% O2) released less H2O2 (29.6 +/- 1.3% and 4.2 +/- 0.7%, respectively) compared with EC exposed to normoxia (20% O2). The extracellular release of H2O2 from EC previously exposed to hypoxia for 24 h increased immediately after reoxygenation (20% O2) to 272 +/- 48%, as compared with EC exposed continuously to normoxia (100% release). Inhibition of xanthine oxidase (XO) by allopurinol did not reduce the release of H2O2 from cells exposed to normoxia or hypoxia followed by reoxygenation. Furthermore, inhibitors of cyclooxygenase (indomethacin), phospholipase A2 (quinacrine and chlorpromazine), nitric oxide synthase (L-arginine analogs), the mitochondrial electron transport chain (rotenone and cyanide), and cytochrome P-450 (methoxypsoralen) had no or minimal effect on this release. On the other hand, inhibitors of protein kinase C (calphostin and staurosporine) and NADPH oxidase (diphenyliodonium) reduced the release of H2O2 from EC in a dose-dependent manner in both exposure groups. In separate experiments, plasma membranes isolated from EC were found to produce H2O2 in the presence of NADH or NADPH as electron donors. This was inhibited by diphenyliodonium but not by allopurinol.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of endothelial cell xanthine dehydrogenase xanthine oxidase gene expression by oxygen tension.

Recent studies have demonstrated that xanthine dehydrogenase/xanthine oxidase (XD/XO) activities of bovine endothelial cells (EC) are inversely regulated by O2 tensions to which the cells are exposed. We have confirmed these reports and extended the observation to a variety of cells from other sources. All EC that had detectable XD/XO activity demonstrated the greatest activity at the lowest O2 level. Bovine pulmonary artery smooth muscle cells showed XD/XO activity only under hypoxic conditions. The ratio of XO to XO+XD did not change significantly under various O2 concentrations for all cell types tested. Treatment of bovine pulmonary artery and rat epididymal fat pad EC with actinomycin D (1 microgram/ml), an inhibitor of transcription, suppressed XO and XO+XD activities in cells exposed both to 20 and 3% O2. High-dose cycloheximide (5 micrograms/ml), an inhibitor of translation, also reduced XO and XO+XD activities in these cells, whereas low-dose cycloheximide (0.5 microgram/ml) enhanced the stimulatory effect of hypoxia on XO+XD activity. We developed a digoxigenin-labeled probe that recognizes and hybridizes to rat XD cDNA and used it to examine the effect of O2 concentration on XD/XO mRNA expression of rat epididymal fat pad EC. XD/XO mRNA concentration was increased in cells exposed to hypoxia and decreased in cells exposed to hyperoxia compared with normoxic cells. The increase in mRNA concentration resulting from exposure to hypoxia was enhanced by cycloheximide. There was no change in XD/XO mRNA stability in cells exposed to hypoxia compared with normoxia. We conclude that the regulation of XD/XO by oxygen tension most likely occurs at the transcriptional level.

Enhanced rate of H2O2 release from bovine pulmonary artery endothelial cells induced by TGF-beta 1.

We have previously reported that transforming growth factor-beta 1 (TGF-beta 1) produces a "prooxidant" effect on cultured bovine pulmonary artery endothelial cells (BPAEC) [S. K. Das and B. L. Fanburg. Am. J. Physiol. 261 (Lung Cell. Mol. Physiol. 5): L249-L254, 1991]. This effect was found to be associated with a lowering of total cellular GSH (A.C. White, S. K. Das, and B.L. Fanburg. Am. J. Respir. Cell Mol. Biol. 6: 364-368, 1992). In this study, we demonstrate a twofold increase in the rate of extracellular H2O2 release from BPAEC after a 72-h exposure to TGF-beta 1 (2 ng/ml, added at times 0 and 48 h). Increasing and decreasing the levels of cellular GSH with diethylmaleate (DEM, 0.05 mM) and buthionine sulfoximine (BSO, 0.01 mM), respectively, did not affect the rate of TGF-beta 1-induced increase in H2O2 release when compared with the individual effects of these reagents on control cells. The addition of BSO (0.01 mM) to control cells failed to demonstrate an increase in the rate of H2O2 release, despite a more profound decrease in cellular GSH by these cells than detected in cells treated with TGF-beta 1 alone. Moreover, a single dose of TGF-beta 1 (2 ng/ml) induced a 63-85% increase in the rate of H2O2 release within 16 h of exposure, well before the previously demonstrated lowering of cellular GSH. These results indicate that the increase in H2O2 production by TGF-beta 1-stimulated BPAEC is associated with, but does not appear to be the result of, a lowering of cellular GSH. This study further suggests that the TGF-beta 1-induced H2O2 production occurs at a site inaccessible to detoxification by GSH.