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Keywords:

  • PAI-1;
  • acute phase response;
  • inflammation

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Summary. Background: The plasma levels of the plasminogen activator-inhibitor type 1 (PAI-1) are consistently elevated in patients with sterile tissue injury, often accompanied by a systemic acute phase protein response. It remains unknown, however, whether and to what extent PAI-1 affects the host response to trauma. Methods and results: By using the well-established murine model of turpentine-induced tissue injury we compared local and systemic inflammatory responses in PAI-1 gene-deficient (PAI-1–/–) and normal wild-type (Wt) mice. Subcutaneous turpentine injection elicited strong increases in PAI-1 protein concentration in plasma and at the site of injury, but not in liver. PAI-1 mRNA was locally increased and expressed mainly by macrophages and endothelial cells. PAI-1 deficiency greatly enhanced the early influx of neutrophils to the site of inflammation, which was associated with increased edema and necrosis at 8 h after injection. Furthermore, PAI-1–/– mice showed a reduced early interleukin (IL)-6 induction with subsequently lower acute phase protein levels and a much slower recovery of body weight loss. Conclusion: These findings suggest that PAI-1 is not merely a marker of tissue injury but plays a functional role in the local and systemic host response to trauma.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Local tissue injury is often associated with a systemic inflammatory response, the so-called acute phase response [1]. This non-specific host response to tissue damage is often seen in patients after trauma, major surgery, burn, tissue infarction or during advanced cancer. The subcutaneous injection of turpentine is a well-established murine model to study the acute phase response induced by local tissue damage [2–6]. The inflammatory reaction to turpentine is characterized by local inflammation and abscess formation, fever, cytokine production, changes in acute phase protein levels, loss of body weight and anorexia. The cytokines interleukin (IL)-1β and IL-6 play an essential role in initiation of the acute phase response [2–6]. IL-1β-deficient mice as well as mice treated with anti-IL-1 receptor antibodies were not able to mount a normal inflammatory reaction to turpentine-induced local tissue damage as reflected by undetectable IL-6 levels and the absence of weight loss and the acute phase protein response [2,6–8]. Administration of anti-IL-6 antibodies and IL-6 deficiency also caused a dramatic reduction of turpentine-induced acute phase protein expression and weight loss in mice [3,4,9]. Together these data indicate that the acute phase response to sterile tissue injury is initiated by IL-1, and that the subsequently induced IL-6 is the essential mediator of the systemic acute phase response.

Plasminogen activator inhibitor type 1 (PAI-1) is the main physiological inhibitor of both tissue-type and urokinase-type plasminogen activator and thereby plays an important role in regulation of the fibrinolytic system. PAI-1 has also been reported to act as an acute phase protein [10,11] and plasma PAI-1 levels rise markedly during disease states often associated with a sterile acute phase response, including trauma, surgery and burn injury [11–15]. In line, turpentine injection elicits an increase in plasma PAI-1 levels in mice, a response that is absent in IL-1β-deficient mice [16]. Recently, it has become clear that PAI-1 probably has other properties besides its inhibitory role in the fibrinolytic system. In particular, PAI-1 has been suggested to be involved in extracellular matrix proteolysis, cellular adhesion and migration [17,18]. However, the overall knowledge about the role of PAI-1 in inflammatory processes is very limited. Here we used the turpentine-induced abscess model to obtain insight into the role of endogenous PAI-1 in inflammation, considering that this model allows us to determine the function of PAI-1 in both the (systemic) acute phase protein response as well as in the local inflammatory response and the recruitment of neutrophils during the formation of an abscess.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Animals

All experiments were approved by the institutional animal care and use committee of the Academic Medical Center (Amsterdam, the Netherlands). C57BL/6 mice were purchased from the Jackson Laboratories (Bar Arbor, ME, USA). PAI-1-deficient (PAI-1–/–) mice [19] and wild-type (Wt) mice were on a mixed Ola129/C57BL/6 background. Eight-week-old female mice were used for all experiments (eight mice per genotype per time-point). The animals were maintained with a 12-h light/12-h dark cycle and had free access to food and water.

Experimental design

Mice were subcutaneously (s.c) injected with 100 µL of turpentine oil (Sigma, St Louis, MO, USA) or saline into both hind limbs. Mice were weighed 24 h before and at 24-h intervals after turpentine injection for 2 weeks. At different time-points after turpentine or saline injection groups of eight mice were anesthetized by intraperitoneal injection of Hypnorm (Janssen Pharmaceutica, Beerse, Belgium) and midazolam (Roche, Meidrecht, the Netherlands), and blood was taken from the inferior vena cava. Blood was collected in ethylenediamine tetra-acetic acid (EDTA)-coated tubes, spun at 1500 g at 4 °C for 15 min and the supernatants were aliquoted and frozen at – 20 °C until assayed.

Preparation of tissue homogenates

The subcutis and muscle tissue were removed at the injection site of each hind limb, weight and snap-frozen in liquid nitrogen. These frozen specimens were crushed to a powder-like suspension, suspended in 4 volumes of sterile isotonic saline and subsequently lyzed in 1 volume of lysis buffer {300 mmol L−1 NaCl, 15 mmol L−1 Tris [Tris(hydroxymethyl)aminomethane], 2 mmol L−1 MgClH2O, 2 mmol L−1 Triton X-100, pepstatin A, leupeptin, and aprotinine [20 ng mL−1], pH 7.4} on ice for 30 min and spun at 1500 g at 4 °C for 15 min. The supernatant was frozen at – 20 °C. Livers were harvested and homogenized at 4 °C in 4 volumes sterile isotonic saline with a tissue homogenizer (Biospect Products, Bartlesville, OK, USA), lyzed in 1 volume of lysis buffer and spun at 1500 g at 4 °C for 15 min; the supernatant was frozen at −20 °C until assayed.

PAI-1 in situ hybridization

Visualization of PAI-1 mRNA was performed as described [20]. First, 5-µm paraffin sections were mounted on SuperFrost Plus glass slides (Menzel-Gläser, Braunschweig, Germany) and subjected to in situ hybridization with a PAI-1 antisense riboprobe. In vitro transcription of linearized plasmid DNA was performed using [35S]-uridine triphosphate (UTP) (Amersham Pharmacia Biotech, Roosendaal, the Netherlands) to obtain the radiolabeled antisense PAI-1 riboprobe. In situ hybridization was executed by standard procedures. In situ sections were covered with nuclear research emulsion (Ilford Imaging UK, Cheshire, UK), exposed for 3 weeks, and then developed and counterstained with hematoxylin and eosin.

Histology

Injected hind limb tissues were removed, postfixed in 10% formaldehyde in phosphate-buffered saline (PBS) for 24 h, dehydrated in increasing concentrations of ethanol followed by xylene, and embedded in paraffin. Sections (4 µm) were cut and stained with hematoxylin and eosin. Semi-quantitative evaluation of tissue histology was performed independently by a pathologist without knowledge of the type of mice and treatment. The histology scores were evaluated by the size of the infiltrate, necrosis and edema. Each was scored separately from 0 to 6, in which 0 = absent and 1–6 ranges from very mild to extremely severe, respectively. Slides were scored in random order. The three scores per section were summarized to the total histology score. To evaluate the amount of collagen fibers formed around the infiltrates, picrosirius red staining of hind limb sections was performed 7 days after turpentine injection. The percentage of area stained was quantified in 10 random nonoverlapping fields of the area around the edge of the abscess (magnification, × 20) from each animal using a computer-assisted image analysis system (Image-pro Plus, MediaCybernetics, Silver Spring, MD, USA).

Assays

Murine PAI-1 antigen levels were measured in plasma and tissue homogenates by enzyme-linked immunosorbent assay (ELISA) as described [20,21]. Levels of myeloperoxidase (MPO) were measured as described elsewhere [22,23]. Macrophage inflammatory protein (MIP)-2, keratinocyte derived chemokine (KC) and IL-1β levels were measured using commercially available ELISA kits (R&D Systems, Abingdon, UK). IL-6 levels were measured using a commercially available cytometric beads array (CBA) multiplex assay (BDBiosciences, San Jose, CA, USA) in accordance with the manufacturer's recommendations. Serum amyloid P (SAP) was measured by a sandwich ELISA, as described previously [24,25]. In short, we used a sheep antimouse SAP as a coating antibody, and a rabbit antimouse SAP as detecting antibody (both Calbiochem-Novabiochem International, San Diego, CA, USA), after which an antirabbit antibody alkaline phosphatase-conjugated (Sigma Chemical Co., St Louis, MO, USA) was added. The assay was developed using p-nitrophenylphosphate; absorption was measured at 405 nm. Serum C3 was also detected by sandwich ELISA using goat antimouse C3 (ICN, Costa Mesa, CA, USA) as coating antibody, and goat antimouse C3c (Nordic, Tilburg, the Netherlands) as detecting antibody. The assay was developed using tetramethyl benzidine and measured at 450 nm. In both ELISAs a standard curve was made by serial dilutions of acute phase mouse serum (Calbiochem-Novabiochem International) with known concentrations of SAP and C3. Haptoglobin was measured using a commercially available colorimetric assay (Tridelta Development Ltd, Co. Wicklow, Ireland).

Statistical analysis

All values are given as means ± SE. Comparisons between groups were analyzed by Mann–Whitney U-test or two-way analysis of variance (anova, using GraphPad Software, Prism version 4.0, San Diego, CA, USA) followed, when statistically significant, by a post hoc Bonferroni test. A P-value of < 0.05 was considered as a significant difference between groups.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

PAI-1 production upon turpentine-induced tissue injury

To evaluate the role of PAI-1 in the sterile acute phase response, we used a well-established murine model of turpentine induced tissue injury and compared inflammatory responses in PAI-1–/– and Wt mice. First, to confirm PAI-1 production in this model, we measured PAI-1 protein levels in C57BL/6 Wt mice at the site of inflammation (hind limb tissue homogenates), in plasma and in liver homogenates at different time-points after turpentine or saline injection. Turpentine injection increased PAI-1 concentrations locally in hind limb tissue homogenates (P < 0.05 vs. saline; Fig. 1A), as well as systemically in plasma (P < 0.05 vs. saline; Fig. 1B), but not in liver homogenates (Fig. 1C).

image

Figure 1. PAI-1 protein levels. PAI-1 protein concentrations in (A) hind limb homogenates, (B) plasma and (C) liver homogenates measured at different time-points after turpentine (black circles) or saline (open circles) injection. Data are mean ± SEM; n = 8 per group per time-point. *P < 0.05, **P < 0.001 vs. saline. NS, non-significant.

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To obtain insight into the cellular expression of locally produced PAI-1, we performed in situ hybridization on turpentine-injected hind limb tissues. At 1, 3, 5 and 7 days after turpentine injection strong expression of PAI-1 mRNA was observed. PAI-1 expression was colocalized predominantly with macrophages encapsulating the inflammatory infiltrates, and was also present in local endothelial cells (Fig. 2A–D). This local PAI-1 mRNA expression was most pronounced at 1 day after turpentine injection. Neutrophilic granulocytes did not show any PAI-1 expression. Together these data suggest that the site of local tissue injury is a major source of circulating PAI-1 after turpentine administration.

image

Figure 2. Local PAI-1 mRNA expression. PAI-1 mRNA expression in hind limb tissue by in situ hybridization 1 day after subcutaneous injection of turpentine. (A) Black and white picture showing an overview of a turpentine-induced abscess. (B) Dark field image of the same microscopic field as in (A) showing PAI-1 mRNA expression as white points surrounding the abscess correlating with the encapsulating macrophages. Magnification, × 100. (C) Detail of PAI-1 mRNA expression colocalized with macrophages and (D) endothelial cells. Magnification: × 400 and × 200, respectively.

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Increased local inflammation in PAI-1–/– mice early after turpentine injection

To obtain insight into the composition of the turpentine induced infiltrate, histological evaluation of the inflamed hind limb tissue from Wt and PAI-1–/– mice was performed before and at 8 h and 1, 3, 5 and 7 days after turpentine injection. In both mouse strains turpentine injection elicited a strong inflammatory response, resulting in local abscess formation characterized by a neutrophilic granulocyte infiltrate encapsulated by macrophages and fibroblasts. After 8 h PAI-1–/– mice displayed significantly more local tissue injury than Wt mice, as judged by the total histology scores of the size of the granulocytic inflammatory infiltrate and the amount of necrosis and edema (mean score 4.0 ± 0.5 and 2.0 ± 0.3, respectively, P < 0.01, Fig. 3A, B). Furthermore, at this time-point, MPO activity in hind limb homogenates was higher in the PAI-1–/– mice compared to Wt mice (13.1 ± 1.3 vs. 9.1 ± 0.7 units/g min−1, P < 0.05), also indicating a stronger neutrophilic migratory response.

image

Figure 3. Histopathology of local inflammation. Wt (A, C, E) and PAI-1–/– (B, D, F) mice were injected subcutaneously with 100 µL of turpentine. Hind limb tissues were removed after 8 h (A, B), 1 (C, D) and 5 (E, F) days. At 8 h PAI-1–/– mice displayed significantly more tissue injury with larger inflammatory infiltrates and more necrosis and edema. The inserts show destruction of skeletal muscle tissue by neutrophils, which is clearly more extensive in PAI-1–/– mice compared to Wt mice. At 1 and 5 days the extent and severity of tissue injury and inflammation did not differ between PAI-1–/– and Wt mice. Slides are representative for eight mice per group per time-point. Magnification: A–B × 40, inserts × 400, C–D × 100, E–F × 200.

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Thereafter, the extent and the severity of the inflammation increased in both mouse strains, with maximal histology scores reached after 7 days. At the later time-points, the local inflammatory response did not differ histologically between PAI-1–/– and Wt mice (shown for days 1 and 5 in Fig. 3C–F). Also, MPO activtiy was similar at 7 days after turpentine injection (data not shown).

Because PAI-1 has been implicated to play a role in the formation of fibrosis [26–29], we measured the amount of fibrotic tissue in Wt and PAI-1–/– mice at 7 days after turpentine injection. However, there was no difference between Wt and PAI-1–/– mice in fibrosis formation around the inflammatory infiltrate at 7 days after turpentine injection, as investigated by image analysis of picrosirius red staining (2.8 ± 0.5 vs. 3.6 ± 0.9, respectively, not significant).

Local chemokine response

The mouse CXC chemokines KC and MIP-2 have been implicated in the attraction of neutrophils to the site of inflammation [30]. Therefore, the concentrations of these mediators were measured in hind limb homogenates. In both genotypes KC and MIP-2 levels increased significantly after turpentine injection and peaked at 1 and 3 days, respectively. There were no differences between Wt and PAI-1–/– mice in local MIP-2 or KC levels at any time-point after turpentine injection (data not shown).

Reduced local and systemic IL-6 response in PAI-1–/– mice

IL-1β and IL-6 play a pivotal role in the systemic acute phase response upon local tissue injury [2–6]. Therefore we measured the concentrations of these cytokines in hind limb homogenates derived from the site of inflammation and in plasma at different time-points after turpentine injection. Local IL-1β levels showed a quick rise and peaked after 8 h with no differences between PAI-1–/– and Wt mice (Fig. 4). IL-1β remained undetectable in plasma at all time-points in both mouse strains. Local IL-6 levels also peaked at 8 h; at this time-point IL-6 levels were much lower in PAI-1–/– mice than in Wt mice, although the difference did not reach statistical significance due to a relatively large interindividual variation (Fig. 4). In addition, plasma IL-6 levels were also markedly reduced in PAI-1–/– mice at 8 h after turpentine injection compared to Wt mice (P < 0.05, Fig. 4).

image

Figure 4. Reduced IL-6 levels in PAI-1–/– mice. IL-1β and IL-6 concentrations in hind limb homogenates and IL-6 levels in plasma at t = 0 and 8 h after turpentine injection in Wt and PAI-1–/– mice. Data are means ± SEM; n = 8 per group per time-point. *P < 0.05 vs. Wt.

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Acute phase protein response

Because IL-6 is the essential mediator of the systemic acute phase protein response in this model [3,4,9], we next determined the magnitude of the acute phase protein response in PAI-1–/– and Wt mice (Table 1). Turpentine injection caused a significant increase in all acute phase proteins measured in both mouse strains. In Wt mice SAP and C3 reached a plateau phase after 24 h and started to decline after 3 days, whereas haptoglobin levels peaked after 3 days. In PAI-1–/– mice SAP and C3 levels were significantly lower than in Wt mice at 24 h (both P < 0.05 vs. Wt) and showed a delayed peak at 3 days after injection. However, at day 3 PAI-1–/– mice demonstrated higher C3 peak levels than Wt mice. Haptoglobin levels did not differ between the two mouse strains at any time-point after turpentine injection.

Table 1.  Plasma acute phase protein levels after turpentine injection
Time after injectionSerum amyloid P µg/mLComplement 3 µg/mLHaptoglobin mg/mL
WtPAI-1 –/–WtPAI-1 –/–WtPAI-1 –/–
  1. Data are means ± SEM (n = 8 mice per group at each time-point) before and at 8 h and 1, 3, 5 and 7 days after turpentine injection. *P < 0.05 vs. Wt. ND, not detectable.

0 h39 ± 0.940 ± 0.2340 ± 70375 ± 47NDND
8 h50 ± 5.845 ± 3.7530 ± 49396 ± 390.1 ± 0.10.4 ± 0.4
1 day506 ± 44343 ± 24*703 ± 71396 ± 73*7.3 ± 0.59.8 ± 2.7
3 days540 ± 35615 ± 71744 ± 691008 ± 60*16.3 ± 1.714.1 ± 2.1
5 days209 ± 44269 ± 46342 ± 41371 ± 294.5 ± 0.59.1 ± 1.9
7 days153 ± 35116 ± 31539 ± 45533 ± 302.5 ± 0.95.9 ± 2.4

Prolonged turpentine-induced weight loss in PAI-1–/– mice

Weight loss is a clinically important feature of the acute phase response [1–6,8,9]. To study the role of PAI-1 in turpentine-induced weight loss, we measured the body weight of PAI-1–/– and Wt mice daily for 2 weeks after turpentine injection. Both groups of mice demonstrated considerable weight loss in the first 2 days after turpentine injection. Thereafter Wt mice started to regain weight, recovering to their initial body weight after 5 days. In contrast, PAI-1–/– mice did not recover and were still below their initial body weight 2 weeks after turpentine injection (P < 0.05 vs. Wt; Fig. 5).

image

Figure 5. Increased turpentine-induced weight loss in PAI-1–/– mice. Wt and PAI-1–/– mice were injected with 100 µL of turpentine in both hind limbs. Body weight was measured daily. Data are expressed as means ± SEM; n = 8 per group. P-value represents difference between Wt and PAI-1–/– mice.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

PAI-1 is an acute phase protein, which is markedly induced upon sterile tissue injury in humans and mice. In this study, we investigated the role of increased PAI-1 levels during the turpentine-induced sterile acute phase response in mice. We show here that PAI-1 is upregulated after turpentine injection and is produced at the site of tissue injury, mainly by macrophages and endothelial cells. Endogenously produced PAI-1 influenced the inflammatory reaction to turpentine at various levels. Indeed, PAI-1–/– mice had a more pronounced local inflammatory response at 8 h after turpentine injection, characterized by increased neutrophil influx, edema and necrosis. Furthermore, PAI-1–/– mice showed a reduced early IL-6 induction and subsequently lower acute phase protein levels after turpentine injection. Clinically, the altered inflammatory response in PAI-1–/– mice was associated with a much slower recovery of their weight loss.

In an earlier investigation Seki et al. found elevated plasma PAI-1 levels after turpentine injection; these authors also reported increased PAI-1 mRNA in the liver [16]. In contrast, we could not detect an increase in PAI-1 protein levels in liver homogenates. Instead, at the site of injury we found high protein levels of PAI-1 together with clear PAI-1 mRNA upregulation by local macrophages and endothelial cells. Hence, our data suggest that elevated PAI-1 plasma concentrations that accompany a sterile trauma are, at least in part, the result of increased production at the site of tissue injury.

PAI-1 can play a role in the regulation of several physiological and pathological processes implicated in inflammation. First, PAI-1 is the main inhibitor of urokinase plasminogen activator (uPA) and thereby a strong inhibitor of uPA mediated pericellular plasmin generation [31,32]. Cell-associated plasmin is able to degrade various extra-cellular matrix components, which facilitates cellular migration. Therefore, PAI-1 might be a negative regulator of plasmin-mediated cell migration. Secondly, PAI-1 can influence cellular migration by inhibition of uPA receptor and vitronectin-mediated cell adhesion [31,32]. The uPA system regulates cell migration by an interaction of uPA with its receptor uPAR (CD87); in addition, uPAR can bind vitronectin, a component of the extra cellular matrix and a ligand for integrins. PAI-1 can inhibit cell adhesion and migration by inhibiting the activity of uPAR-bound uPA, and by preventing integrin association to vitronectin. Studies with uPAR–/– mice have emphasized the eminent role of this receptor in leukocyte trafficking. Indeed, uPAR–/– mice displayed a profoundly reduced neutrophil recruitment to the peritoneal cavity after intraperitoneal administration of thioglycollate [33]. However, the in vivo relevance of PAI-1 in cell migration has not been addressed directly thus far. We showed that PAI-1 deficiency facilitated the initial influx of neutrophils in response to turpentine injection. These data provide the first in vivo evidence for a negative role for PAI-1 in neutrophil recruitment to the site of inflammation. However, this difference in local cellular accumulation disappeared after 1 day. Thus, PAI-1 seems to act as a negative regulator of neutrophil recruitment only during the early phase of turpentine-induced inflammation. Conceivably, other mediators, like the CXC chemokines, overcome the inhibitory effects of PAI-1 later. This theory is strengthened by the fact that local PAI-1 concentrations decrease substantially during the later phase of turpentine-induced inflammation and that, on the other hand, local MIP-2 levels stay strongly elevated up to 7 days after turpentine injection.

As well as its influence on cellular migration, PAI-1 also plays a role in extracellular matrix remodelling and the development of fibrosis [26]. PAI-1 can promote the formation of fibrosis by inhibiting plasmin activation and thereby inhibiting proteolysis of various extracellular matrix components, such as type IV collagen, fibronectin, laminin, proteoglycan and fibrin [26]. Indeed, previous studies with PAI −1–/– mice showed reduced fibrosis formation in lungs after bleomycin-induced lung damage [27] as well as in kidneys after ureteral obstruction [28]. Furthermore, PAI-1–/– mice had less fibrotic tissue accumulation after subcutaneous polyvinyl alcohol sponge implantation [29]. During turpentine-induced chronic tissue inflammation an abscess is formed which finally becomes encapsulated with fibrotic tissue. Therefore, we investigated whether there was a difference in the accumulation of collagen fibers around the turpentine-induced inflammatory infiltrate between Wt and PAI-1–/– mice at 7 days after s.c. injection. In contrast to previous studies we could not detect any difference in the formation of fibrotic tissue. This might be explained by the fact that this is a chronic inflammation model and that by the time fibrosis starts to develop PAI-1 levels are already back to baseline.

The induction of IL-1β and IL-6 plays a very important role in this model of sterile inflammation. IL-1β–/– and IL-6–/– mice do not develop an acute phase protein response and do not suffer from weight loss after turpentine injection [4,6]. In the present study, PAI-1–/– mice showed lower local and systemic IL-6 levels early after turpentine injection, whereas the induction of IL-1β, which at least in part is responsible for the IL-6 response [2,6–8], was not different between the genotypes. These data suggest that PAI-1 deficiency reduces the early IL-6 response to turpentine by an IL-1β independent mechanism. Whether PAI-1 can directly influence IL-6 production remains to be established. In any case, as IL-6 is responsible for the liver acute phase protein response in this model [3,4], the lower SAP and C3 levels at 1 day after turpentine injection were probably caused, at least in part, by the reduced IL-6 levels in PAI-1–/– mice at 8 h. In addition, the attenuated IL-6 response might have played a role in the delayed and reduced recovery of body weight in PAI-1–/– mice. Indeed, although IL-6–/– mice did not mount an acute phase response after turpentine injection, they started losing weight at the time that Wt mice were recovering, and IL-6–/– mice even died later [4]. These findings indicate that the IL-6-induced acute phase response is protective and necessary to repair and recover from the localized tissue inflammation. Of note, we did not follow the mice beyond 14 days because at that time-point all animals had already partly recovered from their injury. In addition, due to the lack of sufficient plasma obtained from mice with a turpentine-induced abscess, we were not able to study the metabolic response to injury here.

Notably, several hormones and drugs are known to decrease PAI-1 levels, including estrogen, angiotensin-converting enzyme inhibitors and metformin [34]. Of these, the effect of estrogen on the inflammatory response to subcutaneous turpentine has been investigated previously [35]. In that study, published in 1949, estradiol dipropionate administration to rats of which the adrenals and gonads were removed was associated with the formation of poorly demarcated abscesses with thin walls which were almost completely void of granulation tissue and a fibroblastic response, consisting almost entirely of neutrophils. Although this study is difficult to compare with ours, the combined data suggest that part of the effect of estrogens in this model could be due to diminished PAI-1 levels. The effects of angiotensin-converting enzyme inhibitors and metformin in this model are unknown at present, and further investigations are warranted to determine these.

While our study investigated the role of endogenously produced PAI-1 in the host inflammatory response to a turpentine-induced abscess, the effect of high circulating levels of PAI-1 in this model remains to be established. In order to investigate this, appropriately sustained overexpression of PAI-1 has to be achieved either by using transgenic mice or by administration of a PAI-1-expressing viral vector. Such experiments are of considerable interest and our laboratory is currently examining this important issue.

The current study reveals several novel findings on the role of PAI-1 in inflammation. We show that PAI-1 is produced at the site of inflammation after turpentine injection, where it plays a role in local inflammation by inhibition of early neutrophil influx. Furthermore, PAI-1 deficiency influences the induction of IL-6 and the subsequent acute phase protein response and delays weight loss recovery. Although our data indicate that endogenously produced PAI-1 influences the local and systemic inflammatory host response to tissue injury, the effect of PAI-1 in human inflammation remains to be established.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This work was supported by grant number 2001B114 from the Netherlands Heart Foundation to R. Renckens. Furthermore, we would like to thank Ingvild Kop and Joost Daalhuisen for expert technical assistance.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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