Search tips
Search criteria

Results 1-7 (7)

Clipboard (0)

Select a Filter Below

Year of Publication
Document Types
1.  Bruton’s Tyrosine Kinase Mediates FcγRIIa/Toll-Like Receptor–4 Receptor Crosstalk in Human Neutrophils 
Previous observations by our laboratory indicate that the presence of anti–IL-8 autoantibody:IL-8 immune complexes in lung fluids from patients with acute lung injury/acute respiratory distress syndrome (ALI/ARDS) comprises an important prognostic indicator in the development and ultimate outcome of ALI/ARDS. We also showed that these complexes display proinflammatory activity toward neutrophils through the engagement of FcγRIIa receptors. Because sepsis is one of the most common risk factors for ALI/ARDS, the initial goal of our present study involved investigating the effects of LPS on the expression of FcγRIIa receptors in neutrophils. Our results indicate that LPS triggers an increase in the expression of FcγRIIa on the neutrophil surface, which leads to shortening of the molecular distance between FcγRIIa and Toll-like receptor–4 (TLR4). When such neutrophils are stimulated with anti–IL-8:IL-8 complexes, the TLR4 cascade becomes activated via the engagement of FcγRIIa. The underlying molecular mechanism has been subsequently examined and involves Bruton’s tyrosine kinase (Btk). In conclusion, our study reveals the existence of Btk-dependent molecular cooperation between FcγRIIa and TLR4 signaling cascades in LPS-“primed” human neutrophils. Furthermore, we used fluorescence lifetime imaging to study the interactions between TLR4 and FcγRIIa in human alveolar neutrophils from patients with ALI/ARDS. The results from these experiments confirm the existence of the molecular cooperation between TLR4 and FcγRIIa.
PMCID: PMC3604059  PMID: 23239500
neutrophil; FcуRIIa; TLR4; Btk
2.  Intrapleural Adenoviral Delivery of Human Plasminogen Activator Inhibitor–1 Exacerbates Tetracycline-Induced Pleural Injury in Rabbits 
Elevated concentrations of plasminogen activator inhibitor–1 (PAI-1) are associated with pleural injury, but its effects on pleural organization remain unclear. A method of adenovirus-mediated delivery of genes of interest (expressed under a cytomegalovirus promoter) to rabbit pleura was developed and used with lacZ and human (h) PAI-1. Histology, β-galactosidase staining, Western blotting, enzymatic and immunohistochemical analyses of pleural fluids (PFs), lavages, and pleural mesothelial cells were used to evaluate the efficiency and effects of transduction. Transduction was selective and limited to the pleural mesothelial monolayer. The intrapleural expression of both genes was transient, with their peak expression at 4 to 5 days. On Day 5, hPAI-1 (40–80 and 200–400 nM of active and total hPAI-1 in lavages, respectively) caused no overt pleural injury, effusions, or fibrosis. The adenovirus-mediated delivery of hPAI-1 with subsequent tetracycline-induced pleural injury resulted in a significant exacerbation of the pleural fibrosis observed on Day 5 (P = 0.029 and P = 0.021 versus vehicle and adenoviral control samples, respectively). Intrapleural fibrinolytic therapy (IPFT) with plasminogen activators was effective in both animals overexpressing hPAI-1 and control animals with tetracycline injury alone. An increase in intrapleural active PAI-1 (from 10–15 nM in control animals to 20–40 nM in hPAI-1–overexpressing animals) resulted in the increased formation of PAI-1/plasminogen activator complexes in vivo. The decrease in intrapleural plasminogen-activating activity observed at 10 to 40 minutes after IPFT correlates linearly with the initial concentration of active PAI-1. Therefore, active PAI-1 in PFs affects the outcome of IPFT, and may be both a biomarker of pleural injury and a molecular target for its treatment.
PMCID: PMC3547083  PMID: 23002099
pleural injury; plasminogen activator inhibitor–1; intrapleural fibrinolytic therapy
3.  Lipoprotein Receptor–Related Protein 1 Regulates Collagen 1 Expression, Proteolysis, and Migration in Human Pleural Mesothelial Cells 
The low-density lipoprotein receptor–related protein 1 (LRP-1) binds and can internalize a diverse group of ligands, including members of the fibrinolytic pathway, urokinase plasminogen activator (uPA), and its receptor, uPAR. In this study, we characterized the role of LRP-1 in uPAR processing, collagen synthesis, proteolysis, and migration in pleural mesothelial cells (PMCs). When PMCs were treated with the proinflammatory cytokines TNF-α and IL-1β, LRP-1 significantly decreased at the mRNA and protein levels (70 and 90%, respectively; P < 0.05). Consequently, uPA-mediated uPAR internalization was reduced by 80% in the presence of TNF-α or IL-1β (P < 0.05). In parallel studies, LRP-1 neutralization with receptor-associated protein (RAP) significantly reduced uPA-dependent uPAR internalization and increased uPAR stability in PMCs. LRP-1–deficient cells demonstrated increased uPAR t1/2 versus LRP-1–expressing PMCs. uPA enzymatic activity was also increased in LRP-1–deficient and neutralized cells, and RAP potentiated uPA-dependent migration in PMCs. Collagen expression in PMCs was also induced by uPA, and the effect was potentiated in RAP-treated cells. These studies indicate that TNF-α and IL-1β regulate LRP-1 in PMCs and that LRP-1 thereby contributes to a range of pathophysiologically relevant responses of these cells.
PMCID: PMC3297170  PMID: 22298529
pleural mesothelial cells; uPAR; LRP-1; internalization; half-life
4.  Urokinase Plasminogen Activator Regulates Pulmonary Arterial Contractility and Vascular Permeability in Mice 
The concentration of urokinase plasminogen activator (uPA) is elevated in pathological settings such as acute lung injury, where pulmonary arterial contractility and permeability are disrupted. uPA limits the accretion of fibrin after injury. Here we investigated whether uPA also regulates pulmonary arterial contractility and permeability. Contractility was measured using isolated pulmonary arterial rings. Pulmonary blood flow was measured in vivo by Doppler and pulmonary vascular permeability, according to the extravasation of Evans blue. Our data show that uPA regulates the in vitro pulmonary arterial contractility induced by phenylephrine in a dose-dependent manner through two receptor-dependent pathways, and regulates vascular contractility and permeability in vivo. Physiological concentrations of uPA (≤1 nM) stimulate the contractility of pulmonary arterial rings induced by phenylephrine through the low-density lipoprotein receptor–related protein receptor. The procontractile effect of uPA is independent of its catalytic activity. At pathophysiological concentrations, uPA (20 nM) inhibits contractility and increases vascular permeability. The inhibition of vascular contractility and increase of vascular permeability is mediated through a two-step process that involves docking to N-methyl-d-aspartate receptor–1 (NMDA-R1) on pulmonary vascular smooth muscle cells, and requires catalytic activity. Peptides that specifically inhibit the docking of uPA to NMDA-R, or the uPA variant with a mutated receptor docking site, abolished both the effects of uPA on vascular contractility and permeability, without affecting its catalytic activity. These data show that uPA, at concentrations found under pathological conditions, reduces pulmonary arterial contractility and increases permeability though the activation of NMDA-R1. The selective inhibition of NMDAR-1 activation by uPA can be accomplished without a loss of fibrinolytic activity.
PMCID: PMC3262683  PMID: 21617202
urokinase; NMDA-R; lung; permeability
5.  Neutrophil α-Defensins Cause Lung Injury by Disrupting the Capillary–Epithelial Barrier 
Rationale: The involvement of neutrophil activation in the sentinel, potentially reversible, events in the pathogenesis of acute lung injury (ALI) is only partially understood. α-Defensins are the most abundant proteins secreted by activated human neutrophils, but their contribution to ALI in mouse models is hindered by their absence from murine neutrophils and the inability to study their effects in isolation in other species.
Objectives: To study the role of α-defensins in the pathogenesis of ALI in a clinically relevant setting using mice transgenic for polymorphonuclear leukocyte expression of α-defensins.
Methods: Transgenic mice expressing polymorphonuclear leukocyte α-defensins were generated. ALI was induced by acid aspiration. Pulmonary vascular permeability was studied in vivo using labeled dextran and fibrin deposition. The role of the low-density lipoprotein–related receptor (LRP) in permeability was examined.
Measurements and Main Results: Acid aspiration induced neutrophil migration and release of α-defensins into lung parenchyma and airways. ALI was more severe in α-defensin–expressing mice than in wild-type mice, as determined by inspection, influx of neutrophils into the interstitial space and airways, histological evidence of epithelial injury, interstitial edema, extravascular fibrin deposition, impaired oxygenation, and reduced survival. Within 4 hours of insult, α-defensin–expressing mice showed greater disruption of capillary–epithelial barrier function and ALI that was attenuated by systemic or intratracheal administration of specific inhibitors of the LRP.
Conclusions: α-Defensins mediate ALI through LRP-mediated loss of capillary–epithelial barrier function, suggesting a potential new approach to intervention.
PMCID: PMC2862305  PMID: 20093642
acute lung injury; capillary–epithelial barrier; α-defensins; low-density lipoprotein–related receptor; receptor-associated protein
6.  Anti-Chemokine Autoantibody:Chemokine Immune Complexes Activate Endothelial Cells via IgG Receptors 
Our previous studies revealed that the presence in lung fluids of anti–IL-8 autoantibody:IL-8 immune complexes is an important prognostic indicator for the development and outcome of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). Anti–IL-8:IL-8 complexes purified from lung edema fluids trigger chemotaxis of neutrophils, induce activation of these cells, and regulate their apoptosis, all via IgG receptor, FcγRIIa. Importantly, increased levels of FcγRIIa are present in lungs of patients with ARDS, where FcγRIIa is partially associated with anti–IL-8:IL-8 complexes. In the current study, we demonstrate the ability of anti–IL-8:IL-8 complexes to promote an inflammatory phenotype of human umbilical vein endothelial cells via interaction with FcγRIIa. Human umbilical vein endothelial cells cultured in the presence of the complexes become activated, as shown by increased phosphorylation of ERK, JNK, and Akt, and augmented nuclear translocation of NF-κB. Anti–IL-8:IL-8 complexes also up-regulate expression of intracellular adhesion molecule (ICAM)-1 on the cell surface. Furthermore, we detected increased levels of ICAM-1 on lung endothelial cells from mice in which lung injury was induced by generating immune complexes in alveolar spaces. On the other hand, ICAM-1 expression was unchanged in lungs of γ chain–deficient mice, lacking receptors that interact with immune complexes. Moreover, in lung tissues from patients with ARDS, anti–IL-8:IL-8 complexes were associated with endothelial cells that expressed higher levels of ICAM-1. Our current findings implicate that anti-chemokine autoantibody:chemokine immune complexes, such as IL-8:IL-8 complexes, may contribute to pathogenesis of lung inflammation by inducing activation of endothelial cells through engagement of IgG receptors.
PMCID: PMC2715905  PMID: 19109244
chemokine; autoantibody; lung; IgG receptor
7.  Does activation of the FcγRIIa play a role in the pathogenesis of the acute lung injury/acute respiratory distress syndrome? 
ALI (acute lung injury) and its more severe form ARDS (acute respiratory distress syndrome) are inflammatory diseases of the lung characterized by hypoxaemia and diffuse bilateral infiltrates. Disruption of epithelial integrity and injury to endothelium are contributing factors of the development of ALI/ARDS, and alveolar damage is the most pronounced feature of ALI/ARDS. The resulting increase in lung microvascular permeability promotes influx of inflammatory cells to the alveolar spaces. Oedema fluid contains pro-nflammatory mediators and plasma proteins, including Igs (immunoglobulins). Moreover, several reports describe the presence of autoantibodies and immune complexes [anti-IL-8 (interleukin-8) autoantibody/IL-8 complexes] in lung fluids (oedema and bronchoalveolar lavage fluids) from patients with ALI/ARDS. These immune complexes associate with FcγRIIa (Fcγ IIa receptor) in lungs of patients with ARDS. Furthermore, the expression of FcγRIIa is substantially elevated in lungs of these patients. FcγRIIa appears on virtually all myeloid cells, platelets and endothelial cells. It is a low-affinity receptor for IgG that preferentially binds aggregated immunoglobulins and immune complexes. FcγRs regulate phagocytosis and cell-mediated cytotoxicity, and initiate the release of inflammatory mediators. It should be noted that immune complexes formed between either anti-neutrophil autoantibodies and their specific antigens or anti-HLA (human leucocyte antigen) antibodies and target antigens are implicated in the pathogenesis of TRALI (transfusion-related acute lung injury), and importantly, animal studies indicate that FcγRs are essential for these complexes to cause damage to the lungs. Therefore, we hypothesize that FcγRs such as FcγRIIa could contribute to the pathogenesis of ALI/ARDS.
PMCID: PMC2811426  PMID: 20088831
acute lung injury; FcγRIIa; IgG receptor; lung; signal transduction; ALI, acute lung injury; ARDS, acute respiratory distress syndrome; FcγR, Fcγ receptor; IL, interleukin; ITAM, immunoreceptor tyrosine-based activation motif; ITIM, immunoreceptor tyrosine-based inhibitory motif; KC, keratinocyte-derived chemokine; LIX, lipopolysaccharide-induced CXC chemokine; LPS, lipopolysaccharide; MIP-2, macrophage inflammatory protein 2; TLR4, Toll-like receptor 4; TRALI, transfusion-related acute lung injury

Results 1-7 (7)