Margaret R. Gyetko

Matrix Metalloproteinases Regulate Neovascularization by Acting as Pericellular Fibrinolysins

During angiogenesis, endothelial cells penetrate fibrin barriers via undefined proteolytic mechanisms. We demonstrate that the fibrinolytic plasminogen activator (PA)-plasminogen system is not required for this process, since tissues isolated from PA- or plasminogen-deficient mice successfully neovascularize fibrin gels. By contrast, neovessel formation, in vitro and in vivo, is dependent on fibrinolytic, endothelial cell-derived matrix metalloproteinases (MMP). MMPs directly regulate this process as invasion-incompetent cells penetrate fibrin barriers when transfected with the most potent fibrinolytic metalloproteinase identified in endothelium, membrane type-1 MMP (MT1-MMP). Membrane display of MT1-MMP is required, as invasion-incompetent cells expressing a fibrinolytically active, transmembrane-deleted form of MT1-MMP remain noninvasive. These observations identify a PA-independent fibrinolytic pathway wherein tethered MMPs function as pericellular fibrinolysins during the neovascularization process.

Urokinase receptor-deficient mice have impaired neutrophil recruitment in response to pulmonary Pseudomonas aeruginosa infection.

Leukocytes express both urokinase-type plasminogen activator (uPA) and the urokinase receptor (uPAR, CD87). Evidence in vitro has implicated uPAR as a modulator of β2 integrin function, particularly CR3 (CD11b/CD18, Mac-1). Pseudomonas aeruginosa infection has been demonstrated to recruit neutrophils to the pulmonary parenchyma by a β2 integrin-dependent mechanism. We demonstrate that mice deficient in uPAR (uPAR−/−) have profoundly diminished neutrophil recruitment in response to P. aeruginosa pneumonia compared with wild-type (WT) mice. The requirement for uPAR in neutrophil recruitment is independent of the serine protease uPA, as neutrophil recruitment in uPA−/− mice is indistinguishable from recruitment in WT mice. uPAR−/− mice have impaired clearance of P. aeruginosa compared with WT mice, as demonstrated by CFU and comparative histology. WT mice have diminished neutrophil recruitment to the lung when an anti-CD11b mAb is given before inoculation with the pathogen, while recruitment of uPAR−/− neutrophils is unaffected. We conclude that uPAR is required for the recruitment of neutrophils to the lung in response to P. aeruginosa pneumonia and that this requirement is independent of uPA. Further, we show that uPAR and CR3 act by a common mechanism during neutrophil recruitment to the lung in response to P. aeruginosa. This is the first report of a requirement for uPAR during cellular recruitment in vivo against a clinically relevant pathogen.

The urokinase receptor (CD87) facilitates CD11b/CD18-mediated adhesion of human monocytes.

Urokinase receptors (uPAR; CD87) from complexes with complement receptor 3 (CR3) (CD11b/CD18), a beta2 integrin. In this study, we sought to determine if this association modulates the adhesive function of CR3. Both CR3 and uPAR concentrate at the ventral surface of fibrinogen-adherent human monocytes, and CR3-uPAR coupling increases substantially upon adhesion to fibrinogen. Pretreatment with anti-uPAR monoclonal antibody reduced adhesion to CR3 counterligands (fibrinogen and keyhole limpet hemocyanin) by 50%, but did not affect adhesion to fibronectin, a beta1 integrin counterligand. Antisense (AS) oligonucleotides were used to determine if selectively suppressing uPAR expression also modulates CR3 adhesive function. AS-uPAR oligo reduced CR3-dependent adhesion by 43+/-9% (P<0.01), but did not affect CR3-independent adhesion. To determine if the effects of uPAR are mediated through its ligand, monocytes were pre-treated with AS oligo to block uPA expression. Unlike the effects of blocking uPAR expression, AS-uPA oligo increased adhesion by 46% (P<0.005), and exogenous intact uPA, but not uPA fragments, reversed this effect. We conclude that complex formation with uPAR facilitates the adhesive functions of CR3. This function of uPAR is not dependent upon its occupancy with uPA, which negatively influences adhesion.

Urokinase is required for the pulmonary inflammatory response to Cryptococcus neoformans. A murine transgenic model.

Urokinase (uPA) is hypothesized to provide proteolytic activity enabling inflammatory cells to traverse tissues during recruitment, and it is implicated as a cytokine modulator. Definitive evaluation of these hypotheses in vivo has previously been impossible because uPA could not completely and irreversibly be eliminated. This limitation has been overcome through the development of uPA-deficient transgenic mice (uPA-/-). Using these mice, we evaluated the importance of uPA in the pulmonary inflammatory response to Cryptococcus neoformans (strain 52D). C. neoformans was inoculated into uPA-/- and control mice (uPA+/+), and cell recruitment to the lungs was quantitated. The number of CFU in lung, spleen and brain was determined to assess clearance, and survival curves were generated. By day 21 after inoculation, uPA-/- mice had markedly fewer pulmonary inflammatory (CD45+), CD4+, and CD11b/CD18+ cells compared with uPA+/+ controls (P<0.0007); pulmonary CFUs in the uPA-/- mice continued to increase, whereas CFUs diminished in uPA+/+ mice(P<0.005). In survival studies, only 3/19 uPA+/+ mice died, whereas 15/19 uPA-/- mice died (p<0.001). We have demonstrated that uPA is required for a pulmonary inflammatory response to C. neoformans. Lack of uPA results in inadequate cellular recruitment, uncontrolled infection, and death.

Function of the urokinase receptor (CD87) in neutrophil chemotaxis.

During recruitment, leukocytes respond to chemotaxins and traverse matrix barriers. Urokinase‐type plasminogen activator (uPA), bound to its receptor (uPAR; CD87) facilitates plasmin formation, which promotes matrix proteolysis. Polymorphonuclear leukocytes (PMNs) are critical to the inflammatory response and express both uPA and CD87. To determine whether uPA and CD87 are required for PMN Chemotaxis, PMNs were pretreated with an anti‐CD87 monoclonal antibody (mAb), a neutralizing anti‐uPA mAb, or uPA. PMN Chemotaxis was profoundly suppressed by the anti‐CD87 mAb but was unaffected by anti‐uPA mAb or uPA. The role CD87 plays in Chemotaxis may be related to its ability to associate with CR3. CD87/CR3 coupling can be disrupted by specific saccharides. The same saccharides that disrupt CD87/CR3 coupling (NADC, D‐mannose, and mannoside) inhibit PMN Chemotaxis. We conclude that CD87 plays a crucial role in PMN Chemotaxis in vitro that is independent of uPA enzyme activity but may be related to the ability of CD87 to interact with CR3.

Urokinase-Type Plasminogen Activator Potentiates Lipopolysaccharide-Induced Neutrophil Activation

Urokinase plasminogen activator (uPA) is a serine protease that catalyzes the conversion of plasminogen to plasmin. Although increased circulating levels of uPA are present in endotoxemia and sepsis, conditions in which activated neutrophils contribute to the development of acute organ dysfunction, the ability of uPA to participate directly in LPS-induced neutrophil activation has not been examined. In the present experiments, we show that uPA can enhance activation of neutrophils exposed to submaximal stimulatory doses of LPS. In particular, uPA increased LPS-induced activation of intracellular signaling pathways, including Akt and c-Jun N-terminal kinase, nuclear translocation of the transcriptional regulatory factor NF-κB, and expression of proinflammatory cytokines, including IL-1β, macrophage-inflammatory protein-2, and TNF-α. There was no effect of uPA on LPS-induced activation of p38 mitogen-activated protein kinase in neutrophils. Transgenic mice unable to produce uPA (uPA−/−) were protected from endotoxemia-induced lung injury, as determined by development of lung edema, pulmonary neutrophil accumulation, lung IL-1β, macrophage-inflammatory protein-2, and TNF-α cytokine levels. These results demonstrate that uPA can potentiate LPS-induced neutrophil responses and also suggest that such effects are sufficiently important in vivo to play a major contributory role in neutrophil-mediated inflammatory responses, such as the development of acute lung injury.

New Insights into the Pathogenesis and Treatment of Idiopathic Pulmonary Fibrosis: A Potential Role for Stem Cells in the Lung Parenchyma and Implications for Therapy

Idiopathic Pulmonary Fibrosis (IPF) is a chronic, progressive, and often fatal form of interstitial lung disease. It is characterized by injury with loss of lung epithelial cells and abnormal tissue repair, resulting in replacement of normal functional tissue, abnormal accumulation of fibroblasts and myofibroblasts, deposition of extracellular matrix, and distortion of lung architecture which results in respiratory failure. Despite improvements in the diagnostic approach to IPF and active research in recent years, the molecular mechanisms of the disease remain poorly understood. This highly lethal lung disorder continues to pose major clinical challenges since an effective therapeutic regimen has yet to be identified and developed. For example, a treatment modality has been based on the assumption that IPF is a chronic inflammatory disease, yet most available anti-inflammatory drugs are not effective in treating it. Hence researchers are now focusing on understanding alternative underlying mechanisms involved in the pathogenesis of IPF in the hope of discovering potentially new pharmaceutical targets. This paper will focus on lung tissue repair, regeneration, remodeling, and cell types that may be important to consider in therapeutic interventions and includes a more detailed discussion of the potential targets of current therapeutic attack in pulmonary fibrosis. The discovery that adult bone marrow stem cells can contribute to the formation of differentiated cell types in other tissues, especially after injury, implies that they have the potential to participate in tissue remodeling, and perhaps regeneration. The current promise of the use of adult stem cells for tissue regeneration, and the belief that once irreversibly damaged tissue could be restored to a normal functional capacity using stem cell-based therapy, suggests a novel approach for treatment of diverse chronic diseases. However this optimism is tempered by current evidence that the pathogenesis of pulmonary fibrosis may involve the recruitment of bone marrow-derived fibroblasts, which are the key contributors to the pathogenesis of this chronic progressive disorder. Nevertheless, stem cell-related therapies are widely viewed as promising treatment options for patients suffering from various types of pulmonary diseases. Gender mismatched bone marrow or lung transplant recipients serve as natural populations in which to study the role of bone marrow-derived stem cells in recovery from pulmonary diseases. Understanding the mechanism of recruitment of stem cells to sites of injury, and their involvement in tissue repair, regeneration, and remodeling may offer a novel therapeutic target for developing more effective treatments against this fatal disorder. This article reviews the new concepts in the pathogenesis, current and future treatment options of pulmonary fibrosis, and the recent advances regarding the roles of stem cells in lung tissue repair, regeneration, and remodeling.

Urokinase-Type Plasminogen Activator Is Required for the Generation of a Type 1 Immune Response to Pulmonary Cryptococcus neoformans Infection

Urokinase-type plasminogen activator (uPA)−/− mice cannot mount protective host defenses during infection with the opportunistic yeast Cryptococcus neoformans (52D). Because effective host defense against C. neoformans requires specific immune responses and the generation of type 1 (T1) cytokines, we determined how the absence of uPA impacts these processes. Wild-type (WT) and uPA−/− mice were inoculated with C. neoformans. Macrophage antifungal activity was assessed histologically, T lymphocyte responses in vivo and proliferation in vitro were quantified, and cytokine concentrations were determined by ELISA. uPA−/− macrophages have impaired antimicrobial activity. Regional lymph nodes of infected uPA−/− mice contained fewer cells than WT, suggesting impaired T cell proliferation in response to the pathogen in vivo. In vitro, uPA−/− T lymphocytes had impaired proliferative responses to C. neoformans rechallenge compared with WT. Infected WT mice generated T1 cytokines in the lung, characterized by high levels of IFN-γ and IL-12. uPA−/− mice had decreased levels of IFN-γ and IL-12, and increased IL-5, a type 2 cytokine. In the absence of uPA, the cytokine profile of regional lymph nodes shifted from a T1 pattern characterized by IFN-γ and IL-2 to a weak, nonpolarized response. We conclude that in the absence of uPA, lymphocyte proliferative responses are diminished, and mice fail to generate protective T1 cytokines, resulting in impaired antimicrobial activity. This study provides novel evidence that uPA is a critical modulator of immune responses and of immune cell effector functions in vivo.

Monocyte 1α-hydroxylase regulation: induction by inflammatory cytokines and suppression by dexamethasone and uremia toxin

Abstract: Alveolar macrophages acquire lα‐hydroxylase activity in inflammation, and thereby metabolize 25 hydroxyvitamin D3 (25 D3) to the active metabolite, la,25‐dihydroxyvitamin D3 (1,25 D3, calcitriol). Cal‐ citriol is a potent differentiation agent that modulates mononuclear phagocyte activation and effector functions. The mediators that induce macrophage lα‐hydroxylase activity are not well delineated. Furthermore, it is unclear whether calcitriol is a product only of terminally differentiated macrophages or whether less mature mononuclear phagocytes can produce it as well. The ability of newly recruited monocytes to produce calcitriol as an autocrine differentiation agent is particularly important in inflammation, as it may substantially expand the functional repertoire of these cells. To assess the effects of cytokines on lα‐hydroxylase activity, blood monocytes were cultured in the presence and absence of human recombinant tumor necrosis factor a (TNF‐α), interferon‐7 (IFN‐γ), and interleukins 1 and 2 and then incubated with 25 D3 substrate. The conditioned media were assayed for calcitriol by high‐performance liquid chromatography and competitive receptor binding assay. No detectable calcitriol was produced by unstimulated monocytes. However, all the cytokines markedly increased monocyte calcitriol production (range 133‐151 pg/mg protein; in all cases P < .001). We then determined whether calcitriol production was suppressed by preincubation with either dexamethasone or the putative uremia toxin guanidinosuccinic acid (GSA). Dexamethasone pretreatment significantly inhibited subsequent cytokine‐induced calcitriol production by monocytes, as did GSA (average 69 and 63% of control, respectively).

Endogenously produced urokinase amplifies tumor necrosis factor-alpha secretion by THP-1 mononuclear phagocytes.

This study examined the effects of endogenous urokinase (uPA) on lipopolysaccharide (LPS)‐stimulated tumor necrosis factor α (TNF‐α) secretion in THP‐1 mononuclear phagocytes. Anti‐uPA monoclonal antibody (mAb) suppressed LPS‐driven TNF‐α secretion by 61.6 ± 5.9% (P < .001), and PAI‐1, a uPA inhibitor, suppressed it to 53.1 ± 8.2% of the control value (P < .001). Up‐regulation of TNF‐α mRNA was suppressed in parallel with secreted TNF‐α protein. TNF‐α secretion was unaffected by depleting plasminogen or by aprotinin, a plasmin inhibitor. When endogenous uPA was displaced from the cell, exogenous high‐molecular‐weight (intact) uPA augmented LPS‐driven TNF‐α secretion. By contrast, a uPA fragment containing the catalytic domain was inhibitory, and the uPA receptor‐binding domain had no effect. We conclude that endogenous uPA amplifies TNF‐α neosynthesis of UPS‐stimulated THP‐1 mononuclear phagocytes. The effect requires intact uPA and is independent of plasmin activity. This represents a novel mechanism by which a mononuclear phagocyte–derived protease contributes to generating proinflammatory signals.