Kimberly E. White

Pulmonary Edema Fluid from Patients with Early Lung Injury Stimulates Fibroblast Proliferation through IL-1β-Induced IL-6 Expression

Although the fibroproliferative response to lung injury occurs with a high frequency in patients with clinical acute lung injury, the mechanisms that initiate this response are largely unknown. This study was undertaken first to identify fibroblast mitogenic factors in pulmonary edema fluid, and second to examine the human lung fibroblast’s gene expression profile in response to pulmonary edema fluid. The edema fluid obtained from patients with early lung injury has an eightfold higher concentration of IL-1β and a twofold greater IL-1β-dependent mitogenic effect than does fluid obtained from control patients with hydrostatic pulmonary edema. Furthermore, fibroblasts responded to acute lung injury patient-derived edema fluid through production of soluble mediators that possess an autocrine mitogenic effect. Gene array analysis reveals that acute lung injury edema fluid induces several inflammation-modulating and proliferation-related genes in fibroblasts, whose inductions are similarly dependent on bioactive IL-1β. Most notably, the 20-fold induction of IL-6 mRNA and protein was completely blocked by IL-1 receptor antagonist. The combined addition of IL-1β and IL-6 was mitogenic, and the proliferative response to conditioned medium from IL-1β-exposed cells was blocked by antagonistically acting Abs to IL-6 or to gp130. These novel findings indicate that soluble IL-1β bioactivity and autocrine IL-1β-dependent IL-6 up-regulation are critical initiators of fibroblast activation and proliferation and that they likely play a role in the fibroproliferative response seen in human acute lung injury.

Prostaglandin E2 Mediates IL-1β-Related Fibroblast Mitogenic Effects in Acute Lung Injury through Differential Utilization of Prostanoid Receptors

The fibroproliferative response to acute lung injury (ALI) results in severe, persistent respiratory dysfunction. We have reported that IL-1β is elevated in pulmonary edema fluid in those with ALI and mediates an autocrine-acting, fibroblast mitogenic pathway. In this study, we examine the role of IL-1β-mediated induction of cyclooxygenase-2 and PGE2, and evaluate the significance of individual E prostanoid (EP) receptors in mediating the fibroproliferative effects of IL-1β in ALI. Blocking studies on human lung fibroblasts indicate that IL-1β is the major cyclooxygenase-2 mRNA and PGE2-inducing factor in pulmonary edema fluid and accounts for the differential PGE2 induction noted in samples from ALI patients. Surprisingly, we found that PGE2 produced by IL-1β-stimulated fibroblasts enhances fibroblast proliferation. Further studies revealed that the effect of fibroblast proliferation is biphasic, with the promitogenic effect of PGE2 noted at concentrations close to that detected in pulmonary edema fluid from ALI patients. The suppressive effects of PGE2 were mimicked by the EP2-selective receptor agonist, butaprost, by cAMP activation, and were lost in murine lung fibroblasts that lack EP2. Conversely, the promitogenic effects of mid-range concentrations of PGE2 were mimicked by the EP3-selective agent, sulprostone, by cAMP reduction, and lost upon inhibition of Gi-mediated signaling with pertussis toxin. Taken together, these data demonstrate that PGE2 can stimulate or inhibit fibroblast proliferation at clinically relevant concentrations, via preferential signaling through EP3 or EP2 receptors, respectively. Such mechanisms may drive the fibroproliferative response to ALI.

Urokinase-type Plasminogen Activator Receptor (uPAR) Ligation Induces a Raft-localized Integrin Signaling Switch That Mediates the Hypermotile Phenotype of Fibrotic Fibroblasts

Background: Fibroblasts from patients with idiopathic pulmonary fibrosis (IPF) overexpress the urokinase-type plasminogen activator receptor (uPAR) and are hypermotile. Results: uPAR ligation increases fibroblast motility by localizing α5β1 integrin-Fyn signaling complexes to lipid rafts. Conclusion: The hypermotile phenotype of IPF fibroblasts is due to lipid raft-localized uPAR-integrin-Fyn signaling complexes. Significance: These unique lipid raft signals may be therapeutic targets for IPF. The urokinase-type plasminogen activator receptor (uPAR) is a glycosylphosphatidylinositol-linked membrane protein with no cytosolic domain that localizes to lipid raft microdomains. Our laboratory and others have documented that lung fibroblasts from patients with idiopathic pulmonary fibrosis (IPF) exhibit a hypermotile phenotype. This study was undertaken to elucidate the molecular mechanism whereby uPAR ligation with its cognate ligand, urokinase, induces a motile phenotype in human lung fibroblasts. We found that uPAR ligation with the urokinase receptor binding domain (amino-terminal fragment) leads to enhanced migration of fibroblasts on fibronectin in a protease-independent, lipid raft-dependent manner. Ligation of uPAR with the amino-terminal fragment recruited α5β1 integrin and the acylated form of the Src family kinase, Fyn, to lipid rafts. The biological consequences of this translocation were an increase in fibroblast motility and a switch of the integrin-initiated signal pathway for migration away from the lipid raft-independent focal adhesion kinase pathway and toward a lipid raft-dependent caveolin-Fyn-Shc pathway. Furthermore, an integrin homologous peptide as well as an antibody that competes with β1 for uPAR binding have the ability to block this effect. In addition, its relative insensitivity to cholesterol depletion suggests that the interactions of α5β1 integrin and uPAR drive the translocation of α5β1 integrin-acylated Fyn signaling complexes into lipid rafts upon uPAR ligation through protein-protein interactions. This signal switch is a novel pathway leading to the hypermotile phenotype of IPF patient-derived fibroblasts, seen with uPAR ligation. This uPAR dependent, fibrotic matrix-selective, and profibrotic fibroblast phenotype may be amenable to targeted therapeutics designed to ameliorate IPF.

Urokinase receptor mediates lung fibroblast attachment and migration toward provisional matrix proteins through interaction with multiple integrins.

Fibroblasts from patients with pulmonary fibrosis express higher levels of the receptor for urokinase, and the extent of fibrosis in some animal models exhibits a dependence on the urokinase receptor. Recent observations have identified the urokinase receptor as a trans-interacting receptor with consequences on signaling and cell responses that vary depending on its interacting partner, the relative levels of expression, and the state of cellular transformation. We undertook this study to define the urokinase-type plasminogen activator cellular receptor (u-PAR)-integrin interactions and to determine the functional consequences of such interactions on normal human lung fibroblast attachment and migration. u-PAR colocalizes in lammelipodia/filopodia with relevant integrins that mediate fibroblast attachment and spreading on the provisional matrix proteins vitronectin, fibronectin, and collagens. Inhibitory antibody studies have revealed that human lung fibroblasts utilize alpha(v)beta(5) to attach to vitronectin, predominantly alpha(5)beta(1) (and alpha(v)beta(3)) to attach to fibronectin, and alpha(1)beta(1), alpha(2)beta(1), and alpha(3)beta(1) to attach to collagen. Blocking studies with alpha-integrin subunit decoy peptides and u-PAR neutralizing antibodies indicate that u-PAR modulates the integrin-mediated attachment to purified provisional matrix proteins, to anti-integrin antibodies, or to fibroproliferative lesions from fibrotic lungs. Furthermore, these decoy peptides blunt fibroblast spreading and migration. We show that u-PAR can interact with multiple alpha-integrins but with a preference for alpha(3). Taken together, these data demonstrate that u-PAR may interact with multiple integrins in normal human lung fibroblasts thereby promoting attachment, spreading, and migration. Modulation of fibroblast invasion would be expected to lead to amelioration of fibroproliferative diseases of the lung.

218 ADENOVIRUS IS PROFIBROTIC IN BLEOMYCIN-INDUCED LUNG INJURY IN MICE THROUGH EFFECTS ON ADAPTIVE IMMUNITY

Recent clinical observations suggest that viral infection/reactivation may contribute to the development of idiopathic pulmonary fibrosis in susceptible hosts. Viral gene therapy using adenoviral vectors in the lung is limited by immune response directed against the viral vector and/or its foreign gene. We therefore examined the effects of adenoviral vector (E1, E3 deleted) on the course of bleomycin-induced lung injury/fibrosis in mice. Methods Adenoviral vectors (native and UV-inactivated) expressing luciferase, β-galactosidase, or a human gene were administered intratracheally (IT; 109 PFU/animal) to mice 2 days after bleomycin (4 U/kg IT). Animals were assessed for mortality, lung inflammation (bronchoalveolar lavage [BAL] cell counts and differential), vascular permeability (BAL protein levels) and fibrosis (lung hydroxyproline) over the next 28 days. Results Combined bleomycin/adenoviral vector administration caused a 3-fold increase in 28 day mortality compared with bleomycin alone (20% to 60%; n = 20/group). The excess mortality was completely abrogated by substituting a UV-inactivated vector and partially abrogated with a null-expressing adenoviral vector. Similarly, combined bleomycin/adenoviral vector administration caused a 2-fold increase in the increment of lung collagen (hydroxyproline) compared with bleomycin alone (150 ± 18 μg/animal vs 300 ± 22 μg/animal; n = 5/group), which was abrogated by substituting a UV-inactivated, or a null-expressing adenoviral vector. Neither IT adenoviral vectors alone nor IT saline alone induced lung fibrosis (by hydroxyproline and trichrome stain). Combined bleomycin/adenoviral vector administration also resulted in increased inflammation (2-fold increment in total BAL cells), and alveolar protein levels compared with bleomycin alone, at 10 days after IT bleomycin. Adenoviral vectors alone had no effect on BAL cell counts or protein levels compared with IT saline-administered controls. The alveolar lymphocytosis was also 2-fold higher in the bleomycin/adenovirally administered group compared with the bleomycin alone group. These data indicate that adenoviral vectors are proinflammatory, enhance lymphocytic recruitment into the alveolar compartment and profibrotic. Further, the dependence of these effects on viral/foreign gene expression implicate antiviral adaptive immunity. Viral-induced adaptive immunity may be one mechanism whereby viral infection will induce a fibrotic response in susceptible hosts with underlying lung inflammation.