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Department of Neurology, Klinikum Grosshadern, Ludwig-Maximilians University, Munich, Germany
Tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) have been suggested to play an important role in inflammatory diseases. Increased levels of tPA, uPA, uPA receptor (uPAR), and their inhibitor, plasminogen activator inhibitor (PAI)1, have been found in the cerebrospinal fluid (CSF) of patients with bacterial meningitis. Here, we show that expression of tPA, uPA, uPAR, PAI-1, and PAI-2 is up-regulated during experimental pneumococcal meningitis. In uPAR-deficient mice, CSF pleocytosis was significantly attenuated 24 h after infection, compared with that in infected wild-type (wt) mice. Lack of uPAR did not influence blood-brain barrier permeability, intracranial pressure, expression of chemokines (keratinocyte-derived cytokine and macrophage inflammatory protein2), bacterial killing, or clinical outcome. No differences in pathophysiological alterations were observed in tPA-deficient mice, compared with those in infected wt mice. These results indicate that uPAR participates in the recruitment of leukocytes to the CSF space during pneumoccal meningitis.
Tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) have been suggested to play an important role in inflammatory diseases [1]. Both promote formation of plasmin, a key protease in fibrinolysis and tissue remodeling. Besides its fibrinolytic activity, tPA has been reported to promote anti-inflammatory activities, such as inhibition of the production of reactive oxygen species (ROS) and down-regulation of expression of the proinflammatory cytokines tumor necrosis factor (TNF) and interleukin (IL)1 [2, 3]. In contrast, uPA has been reported to promote proinflammatory activities, such as attraction and activation of leukocytes and facilitation of their adhesion and migration [4, 5]. uPA binds to a specific cellular receptor (uPA receptor ; CD87) that plays a central role in uPA-mediated activation of plasminogen. Besides promoting fibrinolysis and cell migration, uPA mediates cleavage of cell-bound plasminogen to plasmin, which can enhance release of proinflammatory mediators, including cytokines such as IL-1, and activate matrix metalloproteinases (MMPs) [1, 5]. In addition, plasmin has been shown to increase vessel permeability, which can lead to the opening of the blood-brain barrier in the central nervous system (CNS) [6]. The plasminogen activators are regulated by 2 plasminogen activator inhibitors (PAIs), of which PAI-1 is 20100-fold more efficient than PAI-2 [7]. PAI-1 is the main regulator of endogenous fibrinolysis and is a potent inhibitor of activation of MMP [8, 9]. PAI-2 serves as a modulator of monocyte adhesion, proliferation, and differentiation and regulates uPA-dependent degradation of extracellular matrix [10].
Bacterial meningitis is characterized by invasion of leukocytes into the subarachnoidal space, activation of resident cells, and production of cytokines (e.g., IL-1, IL-6, and IL-10), chemokines (e.g., macrophage inflammatory protein (MIP)1, MIP-2, and keratinocyte-derived cytokine [KC]), ROS, and proteases (e.g., MMPs), which cause an inflammatory response that ultimately causes breakdown of the blood-brain barrier and formation of brain edema [11, 12]. These pathophysiological alterations are held responsible for the unfavorable outcome (mortality >24%) of pneumococcal meningitis, which is the most common form of meningitis in adults [12, 13].
In previous studies, we showed that the protein concentrations of uPA, uPAR, and PAI-1 in the cerebrospinal fluid (CSF) were increased in patients with bacterial meningitis, implicating a role in the pathophysiology of the disease [14, 15]. Therefore, we investigated the involvement of the plasminogen activator system in an experimental model of pneumococcal meningitis, using uPAR-deficient (uPAR-/-) and tPA-deficient (tPA-/-) mice.
MATERIALS AND METHODS
Mouse model of pneumococcal meningitis.
All experiments were approved by the Government of Upper Bavaria. Experiments studying the time course of expression of the plasminogen activator system were conducted with wild-type (wt) mice (C57BL/6; Charles River Wiga).
To assess the functional role of tPA and uPAR, tPA-/- and uPAR-/- mice and their respective wt controls were analyzed. Homozygous uPAR-/- mice and their corresponding wt littermates, with a mixed genetic background of 75% C57BL6 and 25% 129 SV/SL, were provided by P. Carmeliet (Flanders Interuniversitary Institute for Biotechnology, Leuven, Belgium). Breeding pairs of tPA-/- mice (on a C57BL/6 background) were purchased from The Jackson Laboratory.
A well-characterized mouse model of pneumococcal meningitis was used, as described elsewhere [16]. In brief, meningitis was induced by transcutaneous injection of 15 L of 107 cfu/mL Streptococcus pneumoniae type 3 into the cisterna magna. Twenty-four hours after infection, mice were clinically evaluated and anesthetized by use of ketamine/xylazine. The clinical score assessed physiological (temperature, weight, tremor, seizure, vigilance, and pilorection) and motor (beam balancing, postural reflex, and paper crunching) parameters and ranged from 0 (no clinical deficits) to 17 points. A catheter was inserted into the cisterna magna, to measure intracranial pressure (ICP) and to determine CSF white blood cell (WBC) counts. Thereafter, mice were deeply anesthetized and perfused with ice-cold PBS. Brains were removed and rapidly frozen.
To determine cerebellar bacterial titers, the cerebellum was removed immediately after the mice were killed; it was homogenized in sterile saline, and serial dilutions were plated on blood agar plates. Only typical pneumoccal cultures were observed on the plates.
Experimental groups.
To study the time course of expression of tPA, uPA, uPAR, PAI-1, and PAI-2, C57BL/6 mice were killed right after intracranial (ic) injection of 15 L of PBS (control) and 4, 8, and 24 h after infection with pneumococci. For each time point, brain samples from 4 individual mice were used and analyzed in duplicate.
Additionally, the following experimental groups were investigated: (1) C57BL/6 mice injected ic with 15 L of PBS (n = 5), (2) C57BL/6 mice injected ic with S. pneumoniae (n = 8), (3) B6.129S2-Plattm1Mlg/J (tPA-/-) mice injected ic with S. pneumoniae (n = 9), (4) C57BL/6 (75%) × 129SV (25%) (uPAR+/+) mice injected ic with 15 L of PBS (n = 5), (5) C57BL/6 × 129SV mice injected ic with S. pneumoniae (n = 10), and (6) uPAR-/- mice injected ic with S. pneumoniae (n = 11).
Immunoassays for murine MIP-2 and KC.
Concentrations of immunoreactive MIP-2 and KC were determined by use of commercially available ELISA kits (Quantikine Assay kits; R&D Systems). In brief, frozen brain sections were homogenized in sample buffer (10 mmol/L HEPES [pH 7.9], 10 mmol/L KCl, 1.5 mmol/L MgCl2, and a mixture of protease inhibitors). The homogenates were centrifuged, and 50 L of the supernatant was used for each determination. Additionally, the protein concentration of the supernatant was measured by use of the Nanoquant assay (Carl Roth). Concentrations of immunoreactive MIP-2 and KC are expressed as picograms per milligrams of total protein.
Measurement of brain albumin content by ELISA.
Brain albumin concentrations, as a marker of blood-brain barrier integrity, were determined as described elsewhere [17]. In brief, Maxisorb plates (Nunc) were coated and incubated with a mouse albuminspecific rabbit polyclonal antibody (Acris). Plates were washed with washing buffer and blocked with blocking buffer. Mouse brain protein extracts diluted in lysis buffer (10 mmol/L HEPES [pH 7.9], 10 mmol/L KCl, 1.5 mmol/L MgCl2, and a mixture of protease inhibitors) were transferred to assigned wells, and plates were incubated for 60 min at room temperature. Bound albumin was detected by use of a goat polyclonal peroxidaseconjugated antimouse albumin antibody, diluted in sample conjugate buffer (50 mmol/L Tris, 0.14 mol/L NaCl, 1% bovine serum albumin, and 0.05% Tween 20 [pH 8.0]) to a concentration of 0.1 g/mL. Plates were incubated for 60 min at room temperature. Enzyme substrate reagent (R&D Systems) was added to the wells, and the wells were incubated for 10 min at room temperature. The colorimetric reaction was stopped by adding 2 mol/L sulfuric acid, and absorbance was read at 450 nm.
Reverse-transcriptase (RT) polymerase chain reaction (PCR).
For determination of expression of tPA, uPA, uPAR, PAI-1, and PAI-2 mRNA, total RNA was prepared from frozen sections by use of Trizol-LS reagent (GIBCO BRL). Oligo(dt)-primed cDNA was prepared from 5 g of total RNA by use of Superscript II RT (GIBCO BRL). Specific primers were designed for -actin (5-GGA CTC CTA TGT GGG TGA CGA GG-3 and 5-GGG AGA GCA ATA GCC CTC GTA AGA T-3 ), tPA (5-CTG AGG TCA CAG TCC AAG CAA TGT-3 and 5GCT CAC GAA GAT GAT GGT GTA AAG A-3 ), uPA (5-TG CCC AAG GAA ATT CCA GGG-3 and 5-GCC AAT CTG CAC ATA GCA CC-3 ), uPAR (5-CAA CAG GAC CAT GAG TTA CCG CAT GG-3 and 5-AGT GGG TGT AGT TGC AAC ACT TCAG-3 ), PAI-1 (5-GTG GTC TTC TCT CCC TAT G-3 and 5-CTC TGA GAA GTC CAC CTG T-3 ), and PAI-2 (5-GAA GAC ACC AAG ATG GTG CT-3 and 5-CAT TCC TGA GAA GTT GGC CT-3 ). After amplification, PCR products were separated on a 1.7% agarose gel and stained with ethidium bromide. Photographs were scanned and analyzed by densitometry.
Statistical analysis.
All values are expressed as mean ± SD. Data sets were compared by use of the unpaired Student's t test. Differences were considered to be significant at P < .05.
RESULTS
Up-regulation of the plasminogen system in the brain during pneumococcal meningitis.
The time course of expression of tPA, uPA, uPAR, PAI-1, and PAI-2 mRNA in the brain was investigated in infected wt mice. Expression of tPA was significantly up-regulated, by 45%, as soon as 4 h after infection, with a further increase (80% above basal level) 24 h after infection (figure 1A). Expression of uPA mRNA was slightly increased (24% above basal level; P < .05) 4 h after infection and showed no significant increase at later time points (figure 1B). Expression of uPAR mRNA was increased >7-fold 4 h after infection and almost 10-fold 24 h after infection (figure 1C).
Pneumococcal infections also caused an increase in expression of the PAIs: within 4 h after infection, PAI-2 mRNA content immediately increased by 470% from basal level, and, 24 h after infection, it increased by 15-fold (figure 1E). Increase in PAI-1 mRNA content was delayed, with no significant increase 4 or 8 h after infection but with an increase 24 h after infection (530% from basal level; P < .05) (figure 1D).
No effect of lack of tPA on pathophysiological alterations during pneumococcal meningitis.
In wt mice, meningitis caused an increase in CSF WBC counts (10,295 ± 3826 WBCs/L), brain albumin content (63 ± 54 ng/g), and ICP (17.5 ± 6.8 mm Hg), which were significantly higher than those in PBS-injected controls (237 ± 114 WBCs/L, 9 ± 2 ng/g, and 1.4 ± 0.6 mm Hg, respectively) (figure 2A2C). Meningitis was associated with a worse clinical status, as determined by the increase in clinical score (13.6 ± 4.5 vs. 0.2 ± 0.4 in controls; P < .05) (figure 2D). Infection with pneumococci also caused a significant increase in cerebral concentrations of KC and MIP-2 (6 ± 9 vs. 496 ± 340 pg/mg and 2 ± 1 vs. 230 ± 303 pg/mg, respectively) (figure 3A).
Induction of meningitis in tPA-/- mice caused an increase in CSF WBC counts (10,625 ± 3779 WBCs/L), brain albumin content (60 ± 58 ng/g), and ICP (14.8 ± 4.0 mm Hg), which were significantly higher than those in uninfected wt mice but were not significantly different from those in infected wt mice (figure 2A2C). There were no significant differences between infected wt and tPA-/- mice with respect to clinical score (13.6 ± 4.5 vs. 12.0 ± 3.7, respectively) (figure 2D) and cerebral levels of chemokines (KC, 512 ± 377 vs. 496 ± 340 pg/mg, respectively; MIP-2, 370 ± 270 vs. 230 ± 303 pg/mg, respectively) (figure 3A). Likewise, lack of tPA did not affect host defense: cerebellar bacterial titers in tPA-/- mice were not significantly different from those in wt mice (9.97 ± 0.84 vs. 9.11 ± 1.0 log cfu/organ, respectively) (figure 4).
Effect of lack of uPAR on meningitis-induced CSF pleocytosis.
As described above, expression of uPA mRNA was only slightly and transiently increased in infected mice, whereas expression of uPAR mRNA was strongly up-regulated throughout the observation period. Therefore, the impact of lack of uPAR on the course of the disease was investigated. In uPAR+/+ mice, intracisternal injection of pneumococci caused an increase in CSF WBC counts (24,675 ± 9181 WBCs/L), brain albumin content (58 ± 71 ng/g), and ICP (17.7 ± 4.8 mm Hg), which were significantly higher than those in uninfected controls (223 ± 110 WBCs/L, 9 ± 2 ng/g, and 1.3 ± 0.5 mm Hg, respectively) (figure 5A3C). Likewise, meningitis caused a significant increase in clinical score (11.3 ± 3.4 vs. 0.2 ± 0.2 in uninfected controls; P < .05) (figure 5D) and cerebral concentrations of KC and MIP-2 (5 ± 8 vs. 917 ± 668 pg/mg and 2 ± 2 vs. 502 ± 460 pg/mg, respectively; P < .05) (figure 3B).
In uPAR-/- mice, meningitis caused an increase in CSF WBC counts (13,259 ± 3960 WBCs/L), which were significantly higher than those in PBS-injected controls but were significantly lower than those in infected wt mice (figure 5A). A decrease in CSF pleocytosis was not associated with a decrease in ICP (14.9 ± 3.9 mm Hg), brain albumin content (47 ± 36 ng/g), or clinical score (10.1 ± 3.5) (figure 5B5D). There were no significant differences in cerebral concentrations of KC and MIP-2 between infected wt and infected uPAR-/- mice (1205 ± 576 vs. 629 ± 356 pg/mg, respectively) (figure 3B). Lack of uPAR did not affect bacterial clearance: cerebellar titers in uPAR-/- mice were not significantly different from those in wt mice (9.66 ± 1.29 vs. 9.43 ± 1.20 log cfu/organ, respectively) (figure 4).
DISCUSSION
In recent studies, the plasminogen activator system has been shown to be involved in the pathophysiology of various inflammatory diseases. The invasion of leukocytes into the CSF space is a characteristic of bacterial meningitis and triggers a whole cascade of inflammatory processes within the brain.
Here, we have shown that tPA is not involved in the pathophysiology of pneumococcal meningitis. Although cerebral expression of tPA was increased in infected mice, the inflammatory response in tPA-/- mice was not different from that in wt mice, in terms of CSF WBC count, brain albumin content, ICP, expression of chemokines, and clinical score. Likewise, lack of tPA did not influence bacterial clearance. In studies of other inflammatory diseases, the role of tPA has been described as anti-inflammatory, an effect that is attributed in part to its ability to inhibit neutrophil production of ROS [2, 18]. In a mouse model of rheumatoid arthritis, it has been shown that the inflammatory reaction was aggravated in tPA-/- mice, with increased infiltration of leukocytes and expression of IL-1 [19]. Similarly, vascular leak was increased in tPA-/- mice with carrageenan-induced footpad edema [20]. The anti-inflammatory property of tPA was confirmed by application of exogenous tPA in a rat model of acute lung injury resembling acute respiratory stress syndrome in humans [2]. Therefore, tPA attenuated vascular leak, although neutrophil counts in the lungs were unchanged. There may be several reasons for the failure of tPA to ameliorate the pathophysiologic alterations during pneumococcal meningitis. First, concomitant to the increase in expression of tPA, which was 80% above the basal level, induction of meningitis caused a strong increase in cerebral expression of PAI-1 (5-fold) and PAI-2 (15-fold) 24 h after infection. Therefore, the effect of tPA might have been antagonized. Second, it has been shown that, in the brain, tPA can activate microglia, the immunocompetent cells of the CNS [21]. In experimental allergic encephalomyelitis, tPA-/- mice had a later onset of the disease, with attenuated microglial activation and decreased expression of inducible nitric oxide synthase and TNF-, suggesting that tPA plays a protective role in the CNS [22]. Therefore, it is possible that competing beneficial and detrimental properties of tPA canceled each other out during pneumococcal meningitis in the brain.
Expression of uPA was only slightly and temporarily increased in wt mice during meningitis, indicating an inferior role for uPA during pneumococcal meningitis. In contrast, expression of uPAR increased as soon as 4 h after infection, with an increase of >7-fold, compared with basal levels. In uPAR-/- mice with pneumococcal meningitis, CSF WBC counts were decreased by almost 50%, compared with those in infected wt mice, which indicates that uPAR is involved in the recruitment of leukocytes. Impaired recruitment of leukocytes in uPAR-/- mice has also been observed during other inflammatory diseasesfor example, granulocytic influx was significantly decreased in mice lacking uPAR during pneumococcal pneumonia [23] and thioglycollate-induced peritonitis [24]. uPAR, which lacks its own cytoplasmic domain, transduces signals to the cell interior by forming a complex with transmembrane 2 integrins (CD11b/CD18), thereby facilitating migration of leukocytes [4]. Accordingly, in a rabbit model of bacterial meningitis, treatment with antibodies directed against CD18 effectively blocked the development of leukocytosis in the CSF, which resembles the results of the present study [25]. However, the decrease in CSF WBC counts in uPAR-/- mice was less pronounced than that in rabbits treated with antibodies against 2 integrins [25] or their endothelial counterpart, intracellular adhesion molecule1 [26], indicating that integrin-mediated migration of leukocytes does not necessarily require engagement of uPAR. Although CSF pleocytosis was significantly decreased in uPAR-/- mice, the inflammatory reaction was still pronounced, with >13,000 CSF WBCs/L, unchanged levels of chemokines, and unaffected bacterial killing, indicating a robust host response. This might also explain why lack of uPAR resulted in merely a slight, and not a significant, reduction in brain edema, as measured by ICP and brain albumin content. It has been shown, in other disease models, that lack of uPAR does not influence formation of cerebral edema: in a brain trauma model, disruption of the blood-brain barrier in uPAR-/- mice was not different from that in wt mice [27].
In conclusion, we have found that uPAR participates in the recruitment of leukocytes to the CSF space during pneumococcal meningitis. However, it seems that uPAR is not mandatory for this process and that it does not influence the course of the disease.
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