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HEPARANASE PRETREATMENT ATTENUATES ENDOTOXIN-INDUCED ACUTE LUNG INJURY IN RATS

Bashenko, Yulia*; Ilan, Neta*; Krausz, Michael M.; Vlodavsky, Israel*; Hirsh, Mark I.†‡

doi: 10.1097/shk.0b013e3180311d84
Basic Science Aspects
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A central event of systemic inflammation and septic organ injury is infiltration of tissues with polymorphonuclear neutrophils, likely modulated by the integrity of the extracellular matrix underlying the vascular endothelium. In the present study, the effect of matrix-modifying endoglycosidase (heparanase) on endotoxin (LPS)-induced inflammatory lung injury was investigated in rats. Animals were treated with heparanase or LPS or pretreated with heparanase before LPS injection, and acute lung injury was verified histologically and characterized by analysis of bronchoalveolar lavage fluids. Pretreatment with heparanase attenuated the mortality of animals and preserved the histological structure of the lungs. Furthermore, polymorphonuclear neutrophil accumulation and activation, analyzed by myeloperoxidase release and reactive oxygen species production associated with lung injury, were significantly reduced upon heparanase pretreatment. In addition, heparanase pretreatment elevated the IL-10 levels in the pulmonary compartment. Moreover, results from in vitro experiments have identified monocyte-derived IL-10 as an important mediator used by heparanase to suppress inflammatory reactions. The protective effect of heparanase may indicate a novel therapeutic strategy for sepsis.

*Cancer and Vascular Biology Research Center; and Laboratory for Shock and Trauma Research, the Ruth and Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel; and Department of Surgery/Trauma, University of California, San Diego, School of Medicine, San Diego, California

Received 23 Oct 2006; first review completed 14 Nov 2006; accepted in final form 4 Dec 2006

Address reprint requests to Mark I. Hirsh, MD, PhD, Department of Surgery/Trauma, University of California, San Diego, Medical Center, 200 West Arbor Drive, no. 8236, San Diego, CA 92103-8236. E-mail: mhirsh@ucsd.edu.

This work was supported by grants from the Israel Science Foundation (#532/02); National Cancer Institute, NIH (R01-CA106456); the Israel Cancer Research Fund; and the Rappaport Family Institute Fund.

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INTRODUCTION

An inflammatory response to infection (sepsis) or tissue injury (systemic inflammatory response syndrome [SIRS]) frequently results in acute lung injury (ALI) (1). The central event of septic ALI is the infiltration of recruited and primed/activated polymorphonuclear leukocytes (polymorphonuclear neutrophils [PMNs]) into the lung tissue, initiating tissue damage and organ dysfunction (2, 3).

The earliest stage in this process is the extravasation of PMNs from the pulmonary vasculature, initiated by PMN adhesion to the vascular endothelium, followed by transmigration between neighboring endothelial cells, degradation of the subendothelial basement membrane, and migration into the inflamed foci. The basement membrane is a specialized extracellular matrix (ECM) composed of a variety of proteins and polysaccharides secreted by the cells. Heparan sulfate (HS) proteoglycans are abundant components of the ECM consisting of unbranched repeated disaccharide units attached to a core protein. By binding several major ECM constituents (i.e., laminin, fibronectin, collagen type IV), HS proteoglycans are thought to contribute significantly to ECM self-assembly and integrity (4, 5). Studies examining the mobilization of immune and metastasizing cells revealed that several classes of degradative enzymes, often operating in a sequential manner, are required for efficient degradation and remodeling of the ECM barrier, leading to cellular dissemination (6).

Although intensive research has focused on enzymes capable of degrading protein components of the ECM (7, 8), less attention has been paid to enzymes cleaving glycosaminoglycan side chains. Heparanase is an endo-β-d-glucuronidase capable of cleaving HS side chains at a limited number of sites (9, 10). Heparanase activity has been implicated in cancer metastasis, inflammation, neovascularization, and autoimmunity by enhancing the egress of migrating cells from the bloodstream (6, 11-14). Furthermore, heparanase activity can liberate a variety of HS-bound biological mediators, including cytokines and chemokines such as interferon γ, MIP-1β, RANTES, and interleukins (14, 15), thus contributing to regulation of the immune response and inflammation.

Recently, we have demonstrated that exogenous administration of heparanase stimulates Akt-dependent endothelial cell invasion and migration that appear to be independent of heparanase enzymatic activity (16). Similarly, exogenous heparanase was noted to enhance p38 phosphorylation and Src activation, resulting in induced vascular endothelial growth factor and tissue factor expression (17), thus significantly expanding the functional repertoire of this protein. Whereas the activity endogenous heparanase is well implicated in inflammation, the ability of exogenous heparanase to modulate inflammatory reaction has not been examined yet. In the present study, we provide evidence that heparanase pretreatment attenuates ALI induced by endotoxin (LPS) and significantly improves the survival of affected animals.

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MATERIALS AND METHODS

Heparanase

The 65-kd heparanase protein was purified from the culture medium of heparanase-transfected HEK-293 cells, essentially as previously described (16-18).

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Animal experiments

Systemic inflammatory response syndrome and ALI were induced in conscious adult male Sprague-Dawley rats (250-300 g) by an administration of LPS from Escherichia coli O111:B4 (Sigma-Aldrich Israel Ltd., Rehovot, Israel). Rats were fasted overnight before the experiments were conducted but were allowed water ad libitum. Animals were randomly divided into four groups (n = 6): untreated animals (control), rats treated with an i.p. injection of 125 μg · kg−1 heparanase (sham), rats treated with an i.p. injection of 40 mg · kg−1 of LPS (LPS), and rats pretreated with heparanase 60 min before LPS administration (pre-Hepa). Animals in the control and sham groups received a volume of saline equivalent to that of LPS (4 mL · kg−1), whereas LPS-treated rats received a volume of saline equivalent to that of heparanase (4 mL · kg−1). No additional fluid resuscitation was given. The dosage and kind of LPS used for the animal experiments were adopted from those of the study of Brackett et al. (19). Rats were killed 24 h after LPS administration; the lungs were sampled for histological examination and washed, and bronchoalveolar lavage fluids (BALFs) were collected. In separate experiments, rat survival was recorded during 14 days in groups of LPS (n = 20) and pre-Hepa (n = 19). Experiments were performed in accordance with the National Research Council's Guide for the Care and Use of Laboratory Animals, and the protocol was approved by the Animal Care and Use Committee of the Technion Faculty of Medicine.

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Lung histology

The right middle lobes were immersion fixed with phosphate-buffered 4% paraformaldehyde solution (Sigma-Aldrich Israel Ltd.), dehydrated, and embedded in paraffin. Five-micron sections were stained for PMN-specific esterase using naphthol AS-D chloroacetate esterase detection kit (Sigma-Aldrich Israel Ltd.), according to the manufacturer's instructions. Stained lung tissue sections were examined in a blinded fashion for ALI criteria, as previously described (20).

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BALFs and cell collection

Bronchoalveolar lavage fluid was collected essentially as described (21). Briefly, an appropriate polyethylene catheter was inserted into the trachea, and the lungs were filled with 10 mL of warmed phosphate-buffered saline (pH 7.4). The procedure was repeated three times, and BALF samples were combined. Cells were collected by centrifugation, resuspended in phosphate-buffered saline containing 1% fetal calf serum, 3 mmol · L−1 EDTA and 10 mmol · L−1 HEPES (all from Biological Industries, Beth Haemek, Israel), and counted by hemocytometer. Cell viability (>90%) was determined by trypan blue exclusion. Bronchoalveolar lavage fluid cells were identified cytologically by Giemsa-stained cytospin smears. Protein levels in BALF were measured by Bradford reagent (Bio-Rad Laboratories, Munich, Germany), and samples were stored at −80°C until assayed.

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Flow cytometry

Neutrophils were identified using their typical forward- and side-scatter characteristics and gated. Gating of PMNs was verified by staining with fluorescein isothiocyanate-labeled rabbit antirat PMN polyclonal antibody (Accurate Chemical & Scientific Corp., Westbury, NY). Cells (1 × 106 per tube) were incubated with the antibody (0.5 μg; lot E02-F) for 20 min at 4°C, washed, and analyzed by FACSCalibur (Becton Dickinson, Lincoln Park, NJ). In parallel, unstained cells (autofluorescence control) and cells incubated with irrelevant rabbit immunoglobulin G (IgG; Sigma-Aldrich Israel Ltd.) were analyzed.

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PMN functional assays

Analysis of PMN functions was performed essentially as described (22). Briefly, BALF cells (1 × 105 per well) were left untreated or were activated by phorbol-myristate acetate (PMA, 50 ng · mL−1; Sigma-Aldrich Israel Ltd.) to evaluate spontaneous and stimulated production of reactive oxygen species (ROS). Cells were loaded with dihydrorhodamine 123 (2 μg · mL−1; Sigma-Aldrich Israel Ltd.) and incubated at 37°C for 30 min, washed, and examined by flow cytometry. Cells with no dihydrorhodamine added served as control. All measurements were carried out in duplicates and expressed relative to control (ROS production index). Release of PMN azurophilic granules (degranulation) was determined by measurements of myeloperoxidase (MPO) activity in BALF samples or in the PMN supernatant, using the TMB liquid substrate system (Sigma-Aldrich Israel Ltd.) and Ceres UV900 enzyme-linked immunosorbent assay (ELISA) reader. Activity of MPO was calculated as velocity of the optical density raise and normalized for the reaction volume (ΔA650 · min−1 · mL−1) as described (22).

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Cytokine measurement

Concentrations of IL-6 and IL-10 were determined in BALF samples by using ELISA based on CytoSet antibody pairs (Biosource, Nivelles, Belgium), according to the manufacturer's instructions.

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Human blood cells isolation and culture

Peripheral blood mononuclear cells (PBMCs) and PMNs were isolated from peripheral blood of healthy adult donors by centrifugation in Histopaque 1077 and Histopaque 1119 density gradients (Sigma-Aldrich Israel Ltd.). In some experiments, PBMC preparations were depleted of monocytes by adherence to plastic Petri dishes for 45 min at 37°C and 5% CO2 (peripheral blood lymphocytes [PBLs]). Cell fractions were more than 85% pure as assessed by flow cytometry analysis, and cell viability was greater than 97% as determined by propidium iodide (Sigma-Aldrich Israel Ltd.) staining. Isolated cells (1 × 106 per well) were resuspended in 0.5 mL RPMI 1640 medium containing 10% heat-inactivated fetal calf serum and incubated at 37°C in humidified air containing 5% CO2. Cells were left untreated as control, treated with either heparanase (1 μg · mL−1) or LPS (1 μg · mL−1), or treated with heparanase 30 min before treatment with LPS (pre-Hepa). In another series of experiments, PMNs were premixed (1:1) with autologous PBMCs or PBLs and were then treated with heparanase, LPS, or both. Conditioned medium was analyzed for MPO activity, as described above.

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Blocking experiments

To study the role of IL-10 in heparanase-mediated inhibition of PMN activity, autologous PBMCs and PMNs treated with heparanase and LPS were incubated in the absence or presence of 10, 100, and 1,000 ng · mL−1 of neutralizing rabbit antihuman IL-10 polyclonal antibody (PeproTech, Rocky Hill, NJ), and MPO activity in the culture medium was analyzed as described above. As control, cells were treated with an irrelevant rabbit IgG (1 μg · mL−1; Sigma-Aldrich Israel Ltd.).

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Statistics

Data are expressed as mean ± SE. Differences between groups (independent quantitative variables) were evaluated by using one-way ANOVA followed by Newman-Keuls post hoc test. The cumulative survival rates were analyzed by the Kaplan-Meier assay (log-rank pairwise over strata test). Mortality rates were compared using Yates-corrected chi-square test. A value of P < 0.05 was considered statistically significant.

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RESULTS

Heparanase pretreatment improves the survival of LPS-treated rats

To study the involvement of heparanase in inflammatory lung injury, we used LPS-induced model of SIRS in rats. After 24 h of LPS administration, the animals revealed typical symptoms of systemic intoxication: ruffled fur, ocular discharge, hunched posture, decreased activity, diarrhea, and weight loss. At day 14, cumulative survival of rats treated with heparanase before LPS administration was 55% compared with only 20% survival of LPS-treated rats (P = 0.021; Fig. 1).

Fig. 1

Fig. 1

Sepsis-associated multiple organ failure is frequently manifested by ALI, which may progress into life-threatening acute respiratory distress syndrome. Indeed, histological examination of the lungs harvested from LPS-treated rats revealed characteristic abnormalities that included focal circulatory disorders, atelectasis, and major hyperemia with blood congestion (Fig. 2A). Blood capillary bed was occupied by erythrocytes; numerous PMNs were observed within thickened alveolar septae, and several alveolar spaces contained fluid, debris, and inflammatory cells. In contrast, the histological structure of the lungs harvested from rats treated with heparanase before LPS administration was, to a large extent, preserved, and only mild focal hyperemia was detected (Fig. 2B). No thickening of the alveolar septae was observed, and alveolar spaces were free of fluid, debris, or inflammatory cells, suggesting a protective effect of heparanase against LPS-mediated ALI.

Fig. 2

Fig. 2

All survivors were killed after 14 days of survival recording, and their lungs were taken for histological examination. In the lungs of animals that received only LPS, inflammatory signs were detected even after 2 weeks: focal infiltration with PMNs and mononuclear cells, widening and deformation of alveolar walls with collapse of several alveoli, foci of residual hyperemia, and intraalveolar debris. Mean body weight of these animals was 230 ± 2 g, and mean weight gain during 14 days of the experiment was negative: −17.8 ± 2.4 g. In the lungs of rats pretreated with heparanase, no abnormalities were found. Mean body weight was 309 ± 7 g, and mean weight gain was 62.2 ± 4.8 g (P < 0.0001).

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Heparanase pretreatment attenuates PMN accumulation in the lung

To better understand the protective effect of heparanase at the cellular level, BALFs were collected, and cells were harvested by centrifugation. Cytological examination of cell smears revealed that the predominant cells in BALF originating from control and sham rats were macrophages. Polymorphonuclear neutrophil number in BALF of these animals was 0.07 to 0.1 × 105/mL (Fig. 3A), and the ratio of PMNs to macrophages (P/M) was 0.14 ± 0.04 and 0.05 ± 0.03, respectively. The number of inflammatory cells in BALF collected from LPS-treated rats was markedly increased to 8.2 ± 2.4 × 105/mL (P = 0.004), representing a 16.6-fold increase. Most BALF cells were cytologically identified as PMNs, and the P/M ratio increased to 22.08 ± 5.77 (P = 0.016). Pretreatment with heparanase significantly attenuated LPS-induced pulmonary accumulation of PMNs (P = 0.005; Fig. 3A) and reduced the P/M ratio to 3.73 ± 2.07 (P = 0.014).

Fig. 3

Fig. 3

Another parameter indicating the presence of activated PMNs is the activity of MPO measured in BALF, which reflects release of PMN azurophilic granules (degranulation) into the pulmonary milieu. Myeloperoxidase activity in BALF collected from animals of control and sham groups was similar (1.4-1.5 mU · min−1 · mL−1). Endotoxin significantly increased MPO release to 21.9 ± 2.8 mU · min−1 · mL−1 (P = 0.0005), whereas pretreatment with heparanase prevented PMN degranulation and MPO release upon LPS treatment (4.4 ± 1.2 mU · min−1 · mL−1; P = 0.0007).

The differences in inflammatory cell counts and MPO levels corresponded well to the total protein concentrations in BALF (Fig. 3B), reflecting an increase in pulmonary vascular permeability. Thus, protein level in BALF collected from control and sham rats was negligible (Fig. 3B) and was markedly increased upon LPS treatment (0.79 ± 0.26 mg · mL−1; P = 0.021). Pretreatment with heparanase fully prevented this increase (P = 0.019; Fig. 3B).

Cytokines are important regulators of pulmonary inflammatory responses. Levels of typical proinflammatory (IL-6) and anti-inflammatory (IL-10) cytokines in BALF are presented in Table 1. Treatment with heparanase (sham) significantly increased concentrations of both IL-6 and IL-10 in BALF (P < 0.05). Endotoxin, a strong proinflammatory stimulus, expectedly elevated the concentration of IL-6 (P = 0.018) but decreased the level of anti-inflammatory IL-10 (P = 0.019; Table 1). Pretreatment with heparanase prevented LPS-induced rises and falls of IL-6 and IL-10 concentrations, respectively, and kept them close to the level of control animals (Table 1).

Table 1

Table 1

We next determined the functional activity of cells isolated from BALF. Spontaneous ROS production in BALF-isolated cells appeared low and similar among the studied groups (Fig. 4, unstimulated). Stimulation with PMA significantly increased the production of ROS in PMNs isolated from LPS-treated animals (P = 0.0006), whereas pretreatment with heparanase markedly reduced the ROS production (P = 0.0001 vs. LPS; Fig. 4).

Fig. 4

Fig. 4

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Heparanase protective effect is mediated by monocytes and IL-10

Experiments in vivo demonstrated that heparanase might affect PMNs directly or indirectly by eliciting chemical signals (chemokines and cytokines) by other cells. To distinguish between the two possibilities, isolated PMNs were treated in vitro with heparanase or LPS. The effect of treatment was evaluated by MPO release into the culture medium. As shown in Figure 5A, LPS treatment resulted in a significant elevation of MPO release, as expected. Heparanase did not interfere with this effect when applied before LPS (pre-Hepa) and also had no effect on its own (Hepa), suggesting an indirect mechanism of its action on PMNs that included other cells. To identify these cells, isolated PMNs were incubated with LPS and heparanase in the absence or presence of autologous PBMCs or PBMCs depleted of monocytes (PBLs), and MPO activity was evaluated (Fig. 5B). The activity of MPO was significantly reduced upon incubation of PMNs with PBMCs (P < 0.03), whereas monocyte depletion (PBLs) abrogated this effect (Fig. 5B). This result suggests that monocytes are the primary target of heparanase and that monocyte-derived mediators are responsible for PMN attenuation.

Fig. 5

Fig. 5

Because BALF concentrations of anti-inflammatory IL-10 were significantly increased by treatment with heparanase in our in vivo experiments (Table 1), we next incubated PMNs and PBMCs in the presence of increasing concentrations of neutralizing anti-IL-10 antibodies, and MPO activity was evaluated (Fig. 5C). Low MPO activity, reflecting an inhibitory effect of heparanase on PMNs, was found in untreated cells (none) or in cells incubated with control IgG (Fig. 5C). The incubation of cells with anti-IL-10 neutralizing antibody revoked the inhibitory effect of heparanase on PMNs, as reflected by the elevated activity of MPO (P < 0.02; Fig. 5C). This finding suggests that IL-10 mediates, likely coordinating with additional anti-inflammatory mediators, the attenuating effect of heparanase on PMN activity in our experimental model.

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DISCUSSION

In the present study, heparanase pretreatment in vivo attenuated LPS-elicited systemic inflammatory response and ALI by affecting the activation of PMN. We provided evidence that the effect of heparanase on PMNs was indirect, and the inhibition required monocytes and monocytes-derived IL-10.

Release of LPS from the cell wall of gram-negative bacteria frequently initiates sepsis by inducing cascades of metabolic, hemodynamic, and immunological alterations, characterized by hypotension, fever, coagulopathy, organ damage, and death (3, 19). Several clinical and experimental investigations have implicated PMNs in the pathogenesis of septic ALI. Neutrophils, strongly associated with inflammatory lung injury products (ROS and proteases), are abundantly present in BALF of patients with acute respiratory distress syndrome, as well as in BALF samples obtained from experimental animal models of ALI (22-25). Polymorphonuclear neutrophil depletion significantly reduces ALI, whereas restoration of normal PMN blood count exacerbates lung damage (2, 26).

In the present study, beneficial effects of heparanase were observed, manifested by prolonged survival (Fig. 1), well-preserved lung histology, and unaffected pulmonary vascular permeability (Figs. 2 and 3). In light of the pivotal role of PMNs in septic lung injury, the observed favorable effect of heparanase seems to be associated with reduced PMN activation and accumulation in the lung tissue (Figs. 2 and 3). Because heparanase has no inactivating effect on native LPS of E. coli (9), we suggest that the reduced PMN infiltration may be due to decreased PMN activity, as Figure 4 represents. Because our data were acquired using flow cytometry to determine ROS in individual cells and were normalized for number of cells in the sample, the primary protective effect of heparanase in LPS-induced SIRS seems to be suppression of PMN activity, whereas the decrease in number of infiltrating cells is the secondary event. However, we cannot exclude alternative explanations of the observed phenomenon. For example, heparanase can affect PMN accumulation in the lungs by cleavage of glycosaminoglycan side chains, which modifies the biological activities of ECM-associated chemokines and cytokines and therefore disrupts gradient of chemoattractants within the lungs. Akin modulation of chemokine function by HS and subsequent cellular responses have been previously described for IL-8, RANTES, MCP-1, and MIP-1α (27-29). Whether similar mechanism is involved in heparanase-mediated lung protection against LPS is currently under investigation.

Levels of both proinflammatory (IL-6) and anti-inflammatory (IL-10) cytokines were elevated in BALF of animals treated by heparanase (Table 1). This result is fully consistent with the data of others, who have documented the ability of heparanase to release cytokines, chemokines, and growth factors from ECM by cleavage of HS (14, 15). Interestingly, heparanase induced a 13.5-fold increase in IL-10 level, as compared with IL-6. Although the nature of this phenomenon is unknown and should be addressed in further investigations, it suggests a heparanase-induced shift toward anti-inflammatory response in the lungs. In fact, treatment with heparanase before LPS did elicit a substantial increase of IL-10 level in BALF (in 2.4 folds higher than in LPS-treated rats), whereas IL-6 levels were barely detected. This early anti-inflammatory event was later accompanied with complete resolution of ALI signs and prevention of cachexia, suggesting a possible cytokine-dependent mechanism of favorable effects of heparanase pretreatment.

Given the complexity of the in vivo situation, we established an in vitro model, where purified PMNs were exposed to heparanase before LPS addition, and cellular response was monitored by MPO activity. Interestingly, in vitro exposing human PMNs to heparanase before LPS addition had no effect on LPS-mediated MPO release (Fig. 5A), indicating an indirect effect of heparanase. These findings suggest involvement of additional mediator(s), possibly washed away during the cells' isolation. We next focused our efforts on lymphocytes and monocytes, the known regulatory potential of which could support heparanase action in vivo. The ability of PBMCs, but not PBLs, to recapitulate in vitro the inhibitory effect of heparanase on PMN degranulation (Fig. 5B) suggests that this effect is mediated by monocyte-derived factor(s). Interactions between PMNs and monocytes, which are important regulatory immune cells possessing broad range of functional surface receptors and producing numerous cytokines, are well documented (30, 31). Hence, it is not surprising that these cells are involved in heparanase-mediated PMN regulation.

Although monocytes and macrophages secrete a broad spectrum of chemokines and cytokines, a prominent suppressive effect on PMNs is primarily associated with IL-10. This cytokine has been shown to inhibit the production of proinflammatory mediators (IL-1β, TNF-α, and IL-8), block cytokine-induced chemotaxis and oxidative burst, and interfere with PMN-mediated tissue injury (32, 33). In experimental sepsis models, neutralization of IL-10 resulted in exaggerated proinflammatory cytokine expression and animal death, whereas administration of recombinant IL-10 conferred a significant therapeutic protection (34). Similarly, IL-10 levels were markedly elevated in BALF samples collected in our in vivo experiments from rats treated with heparanase alone or when heparanase was applied before LPS. The ability of neutralizing anti-IL-10 antibodies to inhibit the protective effect of PBMCs (Fig. 5C) indicates the important role of this cytokine in heparanase-mediated regulation of PMN functions, ascribed to inflammatory injuring of the host tissues.

In summary, the protective effect of heparanase against ALI suggests a novel function for this protein. Thus, whereas endogenous heparanase activity observed in several blood-born cells appears to be proinflammatory in nature (6, 11-14), exogenously applied heparanase may exert additional effects. These findings suggest that the functions of heparanase are more complex than originally thought. Because in the present study the injected heparanase was detected in BALF of treated animals in both intact and enzymatically processed forms (data not shown), it is not completely clear whether the anti-inflammatory function requires heparanase enzymatic activity. Heparanase has previously been shown to elicit signaling in Akt/PKB, Src, p38, and ERK cascades independently of its enzymatic activity (16-18, 35). Furthermore, constitutive or induced activation of Akt (36) or MAPK (37) has recently been demonstrated to play a critical role in enhanced production of IL-10 by LPS-challenged macrophages. Taken together, these findings suggest that the increased production of IL-10 in our cultured cells can similarly result from signaling cascades activated by administered heparanase. Studies clarifying these issues are currently under way.

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Keywords:

Systemic inflammation; sepsis; acute respiratory distress syndrome; neutrophils

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