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Department of Rheumatology and Inflammation Research, Sahlgrenska University Hospital, Gteborg, Sweden
Urokinase-type plasminogen activator (uPA) is a serine protease that not only displays fibrinolytic function but also modulates innate and adaptive immune responses. In the present study, we assessed whether uPA acts as an endogenous antibiotic. It has been demonstrated that uPA inhibits growth of Staphylococcus aureus both in vivo and in vitro. Importantly, the bactericidal properties of uPA are associated with the serine protease domain of the molecule but are not dependent on its plasminogen-activation potential and cannot be inhibited by plasminogen activator inhibitor type 1 (PAI-1). In a murine infection model, uPA treatment alleviated staphylococcal sepsis by inhibiting bacterial growth. To further evaluate the changes in uPA levels during the course of staphylococcal infection, total uPA and active uPA levels were analyzed in plasma and in kidney homogenates. Expression of total uPA was constant, but PAI-1 levels were dramatically increased in plasma and in kidney homogenates during the course of staphylococcal infection. After infection with staphylococci, the level of metabolically active uPA was unaltered in plasma but was significantly decreased in kidney homogenates. Active uPA levels were inversely related to PAI-1 levels and to bacterial loads in kidney homogenates. In conclusion, we report that uPA acts as an endogenous antibacterial substance that might constitute the first line of host defense against staphylococcal infection. The decreased active uPA levels in infected organs might be due to the dramatically increased PAI-1 production during S. aureus infection.
Urokinase-type plasminogen activator (uPA) is a serine protease that is produced, mainly in the kidneys [1], as a single-chain zymogen (single-chain urokinase) that is essentially inactive [2, 3]. Thereafter, cleavage of single-chain uPA by plasmin or by tumor-associated proteases produces active-form 2-chain urokinase, which is also called high-molecular-weight (HMW) uPA [4, 5]. HMW uPA is composed of a catalytic serine protease domain and a noncatalytic amino-terminal fragment (ATF) that directs the binding of uPA to a specific high-affinity cell-surface receptor, uPAR [6]. uPA catalyzes the cleavage that converts plasminogen to plasmin. This activity is tightly controlled by specific inhibitors, of which plasminogen activator inhibitor type 1 (PAI-1) might be more important than the other 2 inhibitors, PAI-2 and PAI-3, because of its much higher inhibitory efficiency [7, 8]. In addition to its direct PA-inactivating role, PAI-1 promotes the internalization and degradation of uPA through the 2-macroglublin receptor/low-density lipoproteinrelated receptor [9, 10].
The serine protease domain of uPA mediates plasminogen activation on cell surfaces, which plays a pivotal role in cell invasion and tissue remodelling by the degradation of extracellular membrane proteins [11, 12]. Several studies have suggested that uPA is important in the neovascularization, invasion, and metastasis of many solid tumors [1315]. Besides its plasmin-dependent effects, uPA has protease-independent effects, such as mitogenic, migratory, and adhesive properties, that depend on uPA/uPAR binding [1618].
Several lines of evidence indicate that uPA contributes significantly to the pathogenesis of inflammation and immunity. The up-regulation of uPA levels elicited by endotoxin [19] and proinflammatory cytokines, such as interleukin IL-1 and tumor necrosis factor [2022], may represent a mechanism of the innate immune system that is a reaction to infection. uPA potentiates neutrophil activation, including superoxide production [23, 24]. In the absence of the uPA gene, neutrophil activationfor example, phagocytosis, respiratory burst, and degranulationare all impaired [25]. As a consequence, in animal models of pulmonary infection, the deletion of the uPA gene reduces inflammatory cell recruitment and prevents the clearance of bacteria, resulting in uncontrolled infection [2628]. In addition, uPA also participates in adaptive immune responses. Lack of uPA results in impaired T cell activation and proliferation [29]. Furthermore, mice deficient in uPA have a global T cell defect involving polarization of both Th1 and Th2 helper T lymphocytes and are largely immunologically unresponsive [30, 31]. However, the possibility that uPA is an endogenous antibiotic has never been considered in the evaluation of the above data [26, 27].
In the present study, both total uPA levels and active uPA (i.e., PAI-1free uPA) levels were analyzed during staphylococcal infection. Expression of total uPA was constant, but levels of active uPA were significantly decreased in the infected host. PAI-1 levels were strikingly increased in both plasma and kidney homogenates. Because of the negative correlation between active uPA levels and bacterial loads in kidney homogenates, we hypothesized that active uPA might play a role in host protective immunity. Indeed, in vitro, uPA specifically inhibited growth of Staphylococcus aureus. The bactericidal domain within uPA was located in a low-molecular-weight (LMW) portion of the uPA molecule. The bactericidal properties of uPA were independent of its plasminogen-activation activity and were not affected by PAI-1. In the in vivo murine infection model, uPA treatment alleviated staphylococcal sepsis by inhibiting bacterial growth. Thus, we conclude that uPA displays bactericidal properties, which might contribute to host innate immunity during staphylococcal infection.
MATERIAL AND METHODS
Bacterial strains and reagents.
Five S. aureus strains (LS-1 [32], Newman [33], RN6390 [34], P1 [35], and 1061 [36]) were used in the present study. Bacterial strains were handled as described elsewhere [37]. Todd-Hewitt broth (THB) and horse blood agar were obtained from Difco. Human HMW uPA (residues 1411) was purchased from Medac. LMW uPA (residues 136411, containing intact protease domain) and ATF uPA (residues 1135 of the A-chain of uPA) were purchased from American Diagnostica. Bovine serum albumin and dimethyl sulfoxide were purchased from Sigma Chemicals.
Effect of uPA on growth of S. aureus.
A standard number of bacteria (1 × 103 cfu/mL) were incubated in THB at 37°C with increasing concentrations (0200 g/mL) of HMW uPA. At specific intervals, samples of the bacterial mixtures (0.1 mL) were spread on horse blood agar. After incubation for 24 h at 37°C, colonies were counted. To analyze the bactericidal domain of uPA, 100 g/mL HMW uPA (corresponding to 2 mol/L) and an equimolar amount of LMW uPA (63.5 g/mL) or ATF (32.7 g/mL) were incubated with S. aureus LS-1 for 6 h. Results were recorded as the percentage reduction of the bacterial number during different treatment procedures.
Inactivation of uPA.
Three different methods were used to inactivate uPA. (1) Synthetic peptide H-D-Pro-Phe-Arg-chloromethylketone (PPACK) was used to inhibit the serine proteinase activity of uPA. To allow formation of the complex, 200 g/mL uPA was incubated with PPACK, either at an equimolar amount (1.73 g/mL) or at a 10-fold molar excess (17.3 g/mL), in Tris-HCl buffer (pH 7.4) in a 37°C water bath for 30 min. (2) To form the uPA/PAI-1 complex, 200 g/mL HMW uPA was incubated with 60 g/mL PAI-1 in PBS (pH 7.4), for 30 min on ice. (3) HMW uPA (200 g/mL) was denatured by incubating it in a boiling water bath for 30 min. In all cases, intact HMW uPA was used as a positive control.
The inhibitory effects of PPACK, PAI-1, and denaturation on uPA enzymatic activity were evaluated as the hydrolysis of S-2251 (0.3 mmol/L; Chromogenix) in the presence of 300 nmol/L Glu-plasminogen (Biopool). Briefly, uPA cleaves plasminogen to plasmin, which hydrolyzes its chromogenic substrate (H-D-Val-Leu-Lys-pNA/2HCl; S- 2251) to form free pnitroanilin (pNA). The formation of free pNA is proportional to the level of uPA enzymatic activity. All products were then incubated with S. aureus LS-1 for 6 h. The bactericidal effects of uPA and inactivated uPA were analyzed in accordance with the above description.
Infection of mice.
S. aureus LS-1 were thawed, washed in PBS, and then diluted to the appropriate concentration in PBS. Female 68-week-old Naval Medical Research Institute mice were injected intravenously (iv) in the tail vein with 2 different doses of S. aureus LS-1 in 0.2 mL of PBS, as described elsewhere [37]. The study was approved by the Ethics Committee of Sahlgrenska Hospital, and animal-experimentation guidelines were followed.
Experimental infection protocols.
Three sets of experiments were performed by use of S. aureus LS-1. In the first experiment, mice were killed on days 1, 3, and 7 after infection; 7 uninfected mice were used as controls. Plasma and kidney homogenates were obtained for determination of uPA levels. In the second experiment, on day 7, kidney homogenates were extracted aseptically for the assessment of bacterial loads and uPA levels in kidney homogenates. In the third experiment, 8 mice were treated with HMW uPA dissolved in PBS; another 12 mice were treated with the same volume of sterile PBS as was used for controls. The mice were continuously treated with uPA (85 g/g of body weight) intraperitoneally every 24 h until they were killed. Evaluation of body weight development and bacterial load in kidney homogenates was performed.
Bacteriological examination of infected mice.
Both kidneys of each mouse were aseptically removed and homogenized; 100 L of the homogenate was then transferred to horse blood agar plates and incubated for 24 h at 37°C, and the number of colony-forming units was calculated.
Blood samples and kidney homogenates.
In experiments 1 and 2, blood samples from all mice were directly transferred to sodium citrate medium and centrifuged at 800 g for 15 min, for measurement of cytokines. Both kidneys of each mouse were aseptically removed and cut into 2 portions. One portion was used for determination of bacterial load (experiment 2). Another portion was mechanically homogenized and lysed in a 10-fold excess volume of lysis buffer (300 mmol/L NaCl, 15 mmol/L Tris, 2 mmol/L MgCl2, 1% Triton X-100, 3 g/mL aprotinin, and 100 mol/L phenylmethanesulfonyl fluoride [pH 7.4]) for 30 min at 4°C and spun at 1500 g for 15 min at 4°C. This portion was subsequently used for measurement of uPA and PAI-1.
In vitro spleen cell stimulation.
Mouse spleens were aseptically removed and passed through a nylon mesh. Erythrocytes were depleted by lysis in 0.83% ammonium chloride. The resulting single-cell suspension was adjusted to a cell density of 2 × 106 cells/mL in Iscove's complete medium (10% fetal calf serum, 2 mmol/L L-glutamine, 5 × 10-5 mol/L mercaptoethanol, and 50 g/mL gentamicin). Splenocyte cultures were stimulated with toxic shock syndrome toxin (TSST)1 (0.22 g/mL) and peptidoglycans (110 g/mL). After 48 h, supernatants were collected for determination of total PAI-1 levels.
Measurement of active uPA, total uPA, and total PAI-1 levels in plasma and in kidney homogenates from infected mice.
Active murine uPA levels were quantified by use of a uPA activity assay kit (Innovative Research), in accordance with the manufacturer's instructions. Total uPA and total PAI-1 levels were quantified by use of an ELISA kit (Innovative Research), in accordance with the manufacturer's instructions.
Measurement of myeloperoxidase (MPO) levels in kidney homogenates.
MPO, a marker for azurophilic granula, was evaluated by hydrolysis of o-phenylenediamine (0.3 mmol/L; DAKO). Color development was stopped by the addition of 1 N H2SO4. The MPO activity is presented as the absorbance at 492 nm.
Statistical analyses.
The difference in bactericidal killing effect between treated groups, as determined by uPA killing assays, was analyzed by use of the Wilcoxon signed rank test. The changes in active uPA levels, total uPA levels, total PAI-1 levels, and MPO activity in plasma and in kidney homogenates were analyzed on day 7 by use of the Mann-Whitney U test. The severity of arthritis, body weight loss, and bacterial loads in kidney homogenates were assessed by use of the Mann-Whitney U test. The correlations between active/total uPA levels or MPO activity and bacterial loads in kidney homogenates were analyzed by use of Spearman's correlation. The correlation between PAI-1 levels and active uPA levels in kidney homogenates was also analyzed by use of Spearman's correlation. The data are presented as means ± SE. P < .05 was considered to be statistically significant.
RESULTS
Decreased levels of metabolically active uPA during staphylococcal infectioneffect of up-regulation of PAI-1 levels.
Both active uPA levels and total uPA levels in plasma and in kidney homogenates of septic mice were analyzed at specific intervals after bacterial infection, by use of ELISA (figure 1). Active uPA levels in plasma were largely stable during infection, varying from 0.23 to 0.36 ng/mL (figure 1A). They were, however, slightly increased on day 3 (0.23 ± 0.05 vs. 0.35 ± 0.04 ng/mL; P < .05). Intriguingly, active uPA levels in kidney homogenates decreased from 73 ng/mL before infection to 35 ng/mL on day 3 (P < .001) and continued to decrease to 23 ng/mL on day 7 (P < .001) (figure 1B). In contrast, total uPA levels in plasma and in infected kidney homogenates stayed constant throughout the course of infection.
To elucidate why active uPA levels were decreased during infection, PAI-1 was analyzed by use of ELISA. PAI-1 levels in plasma and in kidney homogenates were dramatically increased after infection with S. aureus (figure 2A). PAI-1 levels were detected at low levels in healthy mice (plasma, 2.3 ± 1.2 ng/mL; kidney homogenates, 4.0 ± 0.8 ng/mL). One day after infection, PAI-1 levels were still unaltered. However, on day 3, PAI-1 levels were increased 10-fold in plasma (P < .001) and >50-fold in kidney homogenates (P < .001). On day 7, PAI-1 levels were dramatically increased in both plasma and kidney homogenates (309 ± 295 ng/mL, P < .001 and 7941 ± 5764 ng/mL, P < .001, respectively).
There was a strong inverse correlation between active uPA levels and PAI-1 levels in kidney homogenates throughout the course of infection ( = -0.755; P < .001). These findings indicate that decreased levels of active uPA depend on the inhibitory effect exerted by dramatically increased PAI-1 levels in the infected host.
To assess the triggering mechanisms for the overwhelming increase in PAI-1 levels during staphylococcal infection, we analyzed 2 of the most proinflammatory components of the inoculated bacteria, TSST-1 (a superantigen produced in large amounts by S. aureus LS-1) and peptidoglycans (a vital component of the staphylococcal cell wall). After 48 h of in vitro stimulation with TSST-1, PAI-1 was produced by naive leukocytes in a dose-dependent pattern. In contrast, peptidoglycans did not trigger any PAI-1 production (figure 2B).
Correlation of MPO activity in kidney homogenates with bacterial load and cytokine production during staphylococcal infection.
The enzymatic activity of MPO fluctuated slightly in uninfected mice, as well as in infected mice during the first 3 days after infection (P = .63). In contrast, in infected mice, the enzymatic activity increased >4-fold after 7 days of infection, compared with that in uninfected mice (24 ± 6 vs. 5 ± 0 arbitrary units; P < .01) (figure 3).
The statistical correlations with respect to levels of cytokines, MPO activity, and bacterial loads in kidney homogenates from the mice infected with S. aureus LS-1 for 7 days were calculated. MPO activity strongly correlated with the bacterial loads in kidney homogenates ( = 0.963; P < .01). Indeed, kidney homogenates from 4 mice that did not harbor any bacteria had low MPO activity (4.07.3 U). In another 5 mice, the bacterial loads in kidney homogenates varied from 5 × 106 to 2 × 108 cfu, and the MPO activity was increased accordingly, from 12.4 to 36.6 U. This demonstrates that, after 7 days of staphylococcal infection, infiltration and activation of leukocytes containing azurophilic granula were strongly associated with bacterial accumulation in kidney homogenates. On the contrary, active uPA levels negatively correlated with the bacterial load in kidney homogenates during staphylococcal infection. High bacterial loads (5 × 1062 × 108 cfu) were associated with extremely low levels of active uPA (0.85.5 ng/mL) in kidney homogenates from 5 mice. Bacteria were not found in kidney homogenates of 4 mice. In these cases, high amounts of active uPA (3047 ng/mL) were found in kidney homogenates. In contrast, on day 7, in kidney homogenates, total uPA levels positively correlated with bacterial loads ( = 0.792; P < .05) and PAI-1 levels ( = 0.825; P < .05).
Bactericidal effect of uPA.
To study whether the bactericidal property of HMW uPA is part of its plasminogen-activation capacity, S. aureus LS-1 was incubated with 200 g/mL HMW uPA either alone or precomplexed with PPACK at 2 molar ratios (1 : 1 and 1 : 10). The plasminogen-activation capacity of HMW uPA was almost abrogated after preincubation of HMW uPA precomplexed with PPACK at the 1 : 10 molar ratio, and only 30% of activity remained after preincubation of HMW uPA precomplexed with PPACK at the 1 : 1 molar ratio (table 1). However, HMW uPA precomplexed with PPACK retained its bactericidal properties (uPA/PPACK 1 : 1, 58.9% ± 3.4%; uPA/PPACK 1 : 10, 56.7% ± 4.1%; uPA, 50.9% ± 3.0%; P = .36), demonstrating that the killing of S. aureus by uPA is not mediated by its plasminogen-activation locus.
No effect of PAI-1 on the bactericidal effect of uPA.
Since levels of only active uPA negatively correlated with bacterial loads in kidney homogenates, we hypothesized that only active uPA, and not uPA/PAI-1, kills bacteria. To study this, we preincubated HMW uPA with PAI-1, permitting formation of the complex. Thereafter, the plasminogen-activation properties and the bactericidal function of the HMW uPA/PAI-1 complex were analyzed. Although PAI-1 totally blocked the plasminogen-activation capacity of uPA, the uPA/PAI-1 complex could still kill 60% of bacteria, which is well within the range of intact uPA (55.4% ± 7.7%; P = .39) (figure 5). In contrast, heat denaturation of uPA totally abrogated its ability to activate plasminogen, as well as its ability to kill bacteria. This suggests that the bactericidal effect of uPA is not regulated by PAI-1.
Alleviation of staphylococcal sepsis by HMW uPA treatment.
To investigate the effect of uPA supplementation on staphylococcal sepsis, 8 mice received HMW uPA simultaneously with iv injection of S. aureus (experiment 3) and were continuously treated once daily during the course of sepsis until they were killed. As a control, 12 infected mice received the same volume of PBS. During the course of infection, 1 mouse in the HMW uPA treatment group died, and none of the mice displayed spontaneous bleeding. On day 7, mice in the control group lost 24% (mean) of their body weight; in contrast, the group of mice treated with HMW uPA lost 15% (mean) of their body weight (P = .08) (figure 6A). At the time that the mice were killed, the bacterial load in kidney homogenates of HMW uPAtreated mice was significantly decreased, compared with that in kidney homogenates of control mice (105 × 104 ± 44 × 104 vs. 319 × 104 ± 74 × 104 cfu; P < .05) (figure 6B), demonstrating that extrinsic uPA alleviates staphylococcal sepsis by inhibiting bacterial growth.
DISCUSSION
Traditionally, the biological function of uPA is defined as an enzymatic capacity that permits pericellular proteolysis. In the present study, we have demonstrated that dramatically increased PAI-1 levels during staphylococcal infection led to reduction of metabolically active uPA levels. Moreover, we have reported that uPA kills S. aureus in vitro, and the functional bacteriolytic site of the molecule is located in the vicinity of its serine protease domain. However, the bactericidal property of uPA is independent of its catalytic activity and cannot be controlled by PAI-1. Importantly, uPA treatment alleviated staphylococcal sepsis by inhibiting bacterial accumulation in the internal organs. This is, to our knowledge, the first report of uPA as an endogenous bactericidal substance that acts against staphylococcal infection.
In response to severe infection, which is typically characterized by profound inflammatory drive, the coagulation system becomes activated, resulting in a propensity toward thrombosis [38]. NF-B and mitogen-activated protein kinase transduction pathways activate uPA gene transcription by enhancing phosphorylation and binding of Sp1 protein to the minimal promoter element of the uPA gene [39, 40] and by regulating the methylation state of the uPA gene promoter [41]. Increased total uPA levels in the infected organs have been reported in animal models of other infections [28]. In the present study, total uPA levels in kidney homogenates of S. aureusinfected mice were constant, whereas active uPA levels were down-regulated, and PAI-1 levels were dramatically increased with the development of sepsis. This finding is consistent with the reduced plasminogen-activator activity and elevated PAI-1 levels seen in bronchoalveolar lavage fluid from patients with adult respiratory distress syndrome and pneumonia [4244].
It has been well documented that uPA is required for generation of an adequate inflammatory response. Lack of uPA impedes leukocyte recruitment, resulting in uncontrolled infection and death [26, 27]. On day 7, MPO activity in kidney homogenates was increased, as was the bacterial load, reflecting that, at this time point, infiltration and activation of granulocytes containing azuropilic granula within kidney homogenates had occurred. The decreased levels of active uPA and increased bacterial counts in kidney homogenates coincided with the bactericidal properties of uPA. This might argue in favor of consumption of uPA as a part of bacterial elimination. However, since active uPA and uPA/PAI-1 have identical bactericidal properties, it seems more likely that bacterial influx to kidney homogenates resulted in a more severe inflammation that resulted in higher PAI-1 levels and lower active uPA levels. Indeed, PAI-1 deficiency does not affect the outcome of murine pneumococcal pneumonia [42], suggesting that PAI-1 does not regulate uPA-mediated host defense.
The bactericidal functional site of uPA is located in the LMW portion of uPA (residues 136411), which contains full plasminogen-activation capacity. Could the bactericidal properties of uPA be due to its protease activity PPACK, a synthetic peptide, interacts with the protease active site and totally inhibits uPA activity but does not impede the bactericidal properties of uPA, suggesting that enzymatic activity of uPA is not crucial for the killing of S. aureus. At an inflammatory site, such as the rheumatoid arthritis joints, uPA levels are highly up-regulated [4547]. In the case of S. pneumoniaeinduced pneumonia, uPA levels in the lung homogenates of sick mice were increased from 88 to 200 g/mL [28]; the whole range of levels displayed bactericidal effects in the in vitro system used in the present study. Importantly, because of a potentially uneven distribution of uPA, the local (e.g., intracellular or on the cell surface) level of this molecule might be considerably higher.
The bactericidal function of uPA is of obvious clinical relevance, because uPA is a well-established thrombolytic agent and is extensively used in arterial and venous thromboembolic events. Application of uPA in thrombotic disease might reduce the predisposition for staphylococcal infection. Indeed, the combination of urokinase and appropriate antibiotics has successfully eradicated staphylococcal infection in patients with central venous catheterrelated bacteremia [4850]. This outcome might have been due to the fibrinolytic function of uPA, which releases bacteria trapped in the fibrin matrix and exposes them to systemic antibiotics. However, in line with our results, the bactericidal function of uPA might have also contributed to the control of bacteremia. More importantly, the present study casts light on a possible therapeutic approach in the management of staphylococcal infectionregulation of the expression of endogenous uPA.
Acknowledgments
We thank Margareta Verdrengh and Huamei Fu for excellent technical assistance.
References
1. Kristensen P, Eriksen J, Dano K. Localization of urokinase-type plasminogen activator messenger RNA in the normal mouse by in situ hybridization. J Histochem Cytochem 1991; 39:3419. First citation in article
2. Pannell R, Gurewich V. Activation of plasminogen by single-chain urokinase or by two-chain urokinasea demonstration that single-chain urokinase has a low catalytic activity (pro-urokinase). Blood 1987; 69:226. First citation in article
3. Petersen LC, Lund LR, Nielsen LS, Dano K, Skriver L. One-chain urokinase-type plasminogen activator from human sarcoma cells is a proenzyme with little or no intrinsic activity. J Biol Chem 1988; 263:1118995. First citation in article
4. Stack MS, Johnson DA. Human mast cell tryptase activates single-chain urinary-type plasminogen activator (pro-urokinase). J Biol Chem 1994; 269:94169. First citation in article
5. Kobayashi H, Schmitt M, Goretzki L, et al. Cathepsin B efficiently activates the soluble and the tumor cell receptor-bound form of the proenzyme urokinase-type plasminogen activator (Pro-uPA). J Biol Chem 1991; 266:514752. First citation in article
6. Vassalli JD, Baccino D, Belin D. A cellular binding site for the Mr 55,000 form of the human plasminogen activator, urokinase. J Cell Biol 1985; 100:8692. First citation in article
7. Thorsen S, Philips M, Selmer J, Lecander I, Astedt B. Kinetics of inhibition of tissue-type and urokinase-type plasminogen activator by plasminogen-activator inhibitor type 1 and type 2. Eur J Biochem 1988; 175:339. First citation in article
8. Stump DC, Thienpont M, Collen D. Purification and characterization of a novel inhibitor of urokinase from human urine: quantitation and preliminary characterization in plasma. J Biol Chem 1986; 261:1275966. First citation in article
9. Nykjaer A, Petersen CM, Moller B, et al. Purified alpha 2-macroglobulin receptor/LDL receptor-related protein binds urokinase plasminogen activator inhibitor type-1 complex: evidence that the alpha 2-macroglobulin receptor mediates cellular degradation of urokinase receptor-bound complexes. J Biol Chem 1992; 267:145436. First citation in article
10. Olson D, Pollanen J, Hoyer-Hansen G, et al. Internalization of the urokinase-plasminogen activator inhibitor type-1 complex is mediated by the urokinase receptor. J Biol Chem 1992; 267:912933. First citation in article
11. Ossowski L. Plasminogen activator dependent pathways in the dissemination of human tumor cells in the chick embryo. Cell 1988; 52:3218. First citation in article
12. Tkachuk V, Stepanova V, Little PJ, Bobik A. Regulation and role of urokinase plasminogen activator in vascular remodelling. Clin Exp Pharmacol Physiol 1996; 23:75965. First citation in article
13. Ossowski L, Reich E. Antibodies to plasminogen activator inhibit human tumor metastasis. Cell 1983; 35:6119. First citation in article
14. Duffy MJ, Maguire TM, McDermott EW, O'Higgins N. Urokinase plasminogen activator: a prognostic marker in multiple types of cancer. J Surg Oncol 1999; 71:1305. First citation in article
15. Mazar AP, Henkin J, Goldfarb RH. The urokinase plasminogen activator system in cancer: implications for tumor angiogenesis and metastasis. Angiogenesis 1999; 3:1532. First citation in article
16. Rabbani SA, Mazar AP, Bernier SM, et al. Structural requirements for the growth factor activity of the amino-terminal domain of urokinase. J Biol Chem 1992; 267:141516. First citation in article
17. Waltz DA, Chapman HA. Reversible cellular adhesion to vitronectin linked to urokinase receptor occupancy. J Biol Chem 1994; 269:1474650. First citation in article
18. Busso N, Masur SK, Lazega D, Waxman S, Ossowski L. Induction of cell migration by pro-urokinase binding to its receptor: possible mechanism for signal transduction in human epithelial cells. J Cell Biol 1994; 126:25970. First citation in article
19. Ohta S, Niiya K, Sakuragawa N, Fuse H. Induction of urokinase-type plasminogen activator by lipopolysaccharide in PC-3 human prostatic cancer cells. Thromb Res 2000; 97:3437. First citation in article
20. Marshall BC, Xu QP, Rao NV, Brown BR, Hoidal JR. Pulmonary epithelial cell urokinase-type plasminogen activator. Induction by interleukin-1 beta and tumor necrosis factor-alpha. J Biol Chem 1992; 267:114629. First citation in article
21. Niedbala MJ. Cytokine regulation of endothelial cell extracellular proteolysis. Agents Actions Suppl 1993; 42:17993. First citation in article
22. Takahashi K, Uwabe Y, Sawasaki Y, et al. Increased secretion of urokinase-type plasminogen activator by human lung microvascular endothelial cells. Am J Physiol 1998; 275:L4754. First citation in article
23. Abraham E, Gyetko MR, Kuhn K, et al. Urokinase-type plasminogen activator potentiates lipopolysaccharide-induced neutrophil activation. J Immunol 2003; 170:564451. First citation in article
24. Cao D, Mizukami IF, Garni-Wagner BA, et al. Human urokinase-type plasminogen activator primes neutrophils for superoxide anion release: possible roles of complement receptor type 3 and calcium. J Immunol 1995; 154:181729. First citation in article
25. Gyetko MR, Aizenberg D, Mayo-Bond L. Urokinase-deficient and urokinase receptor-deficient mice have impaired neutrophil antimicrobial activation in vitro. J Leukoc Biol 2004; 76:64856. First citation in article
26. Beck JM, Preston AM, Gyetko MR. Urokinase-type plasminogen activator in inflammatory cell recruitment and host defense against Pneumocystis carinii in mice. Infect Immun 1999; 67:87984. First citation in article
27. Gyetko MR, Chen GH, McDonald RA, et al. Urokinase is required for the pulmonary inflammatory response to Cryptococcus neoformans: a murine transgenic model. J Clin Invest 1996; 97:181826. First citation in article
28. Rijneveld AW, Levi M, Florquin S, Speelman P, Carmeliet P, van Der Poll T. Urokinase receptor is necessary for adequate host defense against pneumococcal pneumonia. J Immunol 2002; 168:350711. First citation in article
29. Gyetko MR, Libre EA, Fuller JA, Chen GH, Toews G. Urokinase is required for T lymphocyte proliferation and activation in vitro. J Lab Clin Med 1999; 133:27488. First citation in article
30. Gyetko MR, Sud S, Chensue SW. Urokinase-deficient mice fail to generate a type 2 immune response following schistosomal antigen challenge. Infect Immun 2004; 72:4617. First citation in article
31. Gyetko MR, Sud S, Chen GH, Fuller JA, Chensue SW, Toews GB. Urokinase-type plasminogen activator is required for the generation of a type 1 immune response to pulmonary Cryptococcus neoformans infection. J Immunol 2002; 168:8019. First citation in article
32. Bremell T, Abdelnour A, Tarkowski A. Histopathological and serological progression of experimental Staphylococcus aureus arthritis. Infect Immun 1992; 60:297685. First citation in article
33. McDevitt D, Francois P, Vaudaux P, Foster TJ. Identification of the ligand-binding domain of the surface-located fibrinogen receptor (clumping factor) of Staphylococcus aureus. Mol Microbiol 1995; 16:895907. First citation in article
34. Novick RP, Ross HF, Projan SJ, Kornblum J, Kreiswirth B, Moghazeh S. Synthesis of staphylococcal virulence factors is controlled by a regulatory RNA molecule. Embo J 1993; 12:396775. First citation in article
35. Sherertz RJ, Carruth WA, Hampton AA, Byron MP, Solomon DD. Efficacy of antibiotic-coated catheters in preventing subcutaneous Staphylococcus aureus infection in rabbits. J Infect Dis 1993; 167:98106. First citation in article
36. Kuusela P, Hilden P, Savolainen K, Vuento M, Lyytikainen O, Vuopio-Varkila J. Rapid detection of methicillin-resistant Staphylococcus aureus strains not identified by slide agglutination tests. J Clin Microbiol 1994; 32:1437. First citation in article
37. Bremell T, Lange S, Yacoub A, Ryden C, Tarkowski A. Experimental Staphylococcus aureus arthritis in mice. Infect Immun 1991; 59:261523. First citation in article
38. Esmon CT. Crosstalk between inflammation and thrombosis. Maturitas 2004; 47:30514. First citation in article
39. Benasciutti E, Pages G, Kenzior O, Folk W, Blasi F, Crippa MP. MAPK and JNK transduction pathways can phosphorylate Sp1 to activate the uPA minimal promoter element and endogenous gene transcription. Blood 2004; 104:25662. First citation in article
40. Das R, Mahabeleshwar GH, Kundu GC. Osteopontin stimulates cell motility and nuclear factor kappaB-mediated secretion of urokinase type plasminogen activator through phosphatidylinositol 3-kinase/Akt signaling pathways in breast cancer cells. J Biol Chem 2003; 278:28593606. First citation in article
41. Pakneshan P, Szyf M, Farias-Eisner R, Rabbani SA. Reversal of the hypomethylation status of urokinase (uPA) promoter blocks breast cancer growth and metastasis. J Biol Chem 2004; 279:3173544. First citation in article
42. Rijneveld AW, Florquin S, Bresser P, et al. Plasminogen activator inhibitor type-1 deficiency does not influence the outcome of murine pneumococcal pneumonia. Blood 2003; 102:9349. First citation in article
43. Bertozzi P, Astedt B, Zenzius L, et al. Depressed bronchoalveolar urokinase activity in patients with adult respiratory distress syndrome. N Engl J Med 1990; 322:8907. First citation in article
44. Idell S, James KK, Levin EG, et al. Local abnormalities in coagulation and fibrinolytic pathways predispose to alveolar fibrin deposition in the adult respiratory distress syndrome. J Clin Invest 1989; 84:695705. First citation in article
45. Belcher C, Fawthrop F, Bunning R, Doherty M. Plasminogen activators and their inhibitors in synovial fluids from normal, osteoarthritis, and rheumatoid arthritis knees. Ann Rheum Dis 1996; 55:2306. First citation in article
46. Brommer EJ, Dooijewaard G, Dijkmans BA, Breedveld FC. Plasminogen activators in synovial fluid and plasma from patients with arthritis. Ann Rheum Dis 1992; 51:9658. First citation in article
47. Jin T, Tarkowski A, Carmeliet P, Bokarewa M. Urokinase, a constitutive component of the inflamed synovial fluid, induces arthritis. Arthritis Res Ther 2003; 5:R917. First citation in article
48. Fishbein JD, Friedman HS, Bennett BB, Falletta JM. Catheter-related sepsis refractory to antibiotics treated successfully with adjunctive urokinase infusion. Pediatr Infect Dis J 1990; 9:6768. First citation in article
49. Ascher DP, Shoupe BA, Maybee D, Fischer GW. Persistent catheter-related bacteremia: clearance with antibiotics and urokinase. J Pediatr Surg 1993; 28:6279. First citation in article
50. Jones GR, Konsler GK, Dunaway RP, Lacey SR, Azizkhan RG. Prospective analysis of urokinase in the treatment of catheter sepsis in pediatric hematology-oncology patients. J Pediatr Surg 1993; 28:3505; discussion 3557. First citation in article