1Department of Molecular Cell Biology and Immunology, 2Department of Nephrology and 3Clinical Chemistry, VU University Medical Center, Amsterdam and 4Department of Experimental Immunohematology, CLB, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
Correspondence and offprint requests to: Jacob van den Born, Department of Molecular Cell Biology and Immunology, VU University Medical Center, FdG, Postbus 7057, 1007 MB Amsterdam, The Netherlands. Email: j.vandenborn{at}vumc.nl
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Abstract |
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Methods. First, we evaluated neutrophil migration in an in vitro transwell model for the peritoneal membrane with monolayers of primary human mesothelial cells (MC) on the lower side and primary human endothelial cells (EC) on top of the same transwell membrane, upon exposure of MC to PD fluid (PDF)-derived components. In addition to this in vitro model, we combined chronic peritoneal exposure to PDF with a peritoneal infection model in the rat. We investigated the kinetics of the chemokine response, neutrophil recruitment and bacterial clearance.
Results. Known chemoattractants, such as fMLP and IL-8, strongly increased neutrophil migration across both cell layers in the in vitro model of the peritoneal membrane. Pre-incubation of the MC layer for 48 h with 55 mM glucose, a combination of two glucose degradation products, methylglyoxal and 3-deoxyglucosone, or conventional dialysis fluid (1:4 dilution), however, did not change the IL-8-induced migration of neutrophils. In concert with this finding we demonstrated an unchanged MC expression of ICAM-1 and VCAM-1 after these pre-treatments. Unexpectedly, chronic i.p. exposure to conventional PDF or a recently developed lactate/bicarbonate-buffered PDF in a rat peritoneal exposure model strongly hampered the chemokine response upon bacterial challenge. Nevertheless, neutrophil recruitment and bacterial clearance were effective and did not differ from rats not pre-exposed to PDF.
Conclusions. We conclude that exposure of MC to PDF does not hamper the recruitment of functional neutrophils upon challenge.
Keywords: chemokine; endothelial cell; mesothelial cell; peritoneal dialysis; rat
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Introduction |
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Chronic treatment with conventional PD fluids (PDF) affects mesothelial cell (MC) function [5]. We and others showed that incubation of cultured MC with glucose, glucose degradation products (GDPs) or advanced glycation end products showed up-regulation of VCAM-1 expression and IL-8 production [6,7]. Intravital microscopy of the mesenteric venules has demonstrated that the initial step in transendothelial migration, the rolling of the leukocytes along the vessel wall is enhanced in rats that had been treated with the conventional PDF [8].
Furthermore, we reported that the ex vivo bacterial killing capacity of the peritoneal cells was highly impaired upon instillation of lactate-buffered PDF in a rat peritoneal exposure model, whereas exposure to bicarbonate/lactate-buffered PDF had only a minor effect [9]. Thus, we have provided strong evidence that peritoneal endothelial cells (EC), MC as well as leukocytes become impaired upon immediate and chronic contact with conventional PDF. The long-term effect of these fluids on the inflammatory response upon bacterial challenge is however less clear.
In the present study we have investigated the effect of dialysis fluids and some of their components on neutrophil migration in vitro and the inflammatory response inducing neutrophil migration upon infection in vivo. We set up an in vitro transwell culture model with primary human MC on the lower side of a porous filter and primary human EC on top of the same filter representing the peritoneal membrane. This enabled us to study neutrophil transmigration in a physiological way, sequentially passing EC and MC monolayers, after exposure of the MC cultures to dialysis fluid, glucose or GDPs. Leukocyte migration across inverted MC monolayers has been shown to be partially dependent on ICAM-1 and on polarized secretion of IL-8 (CXCL-8) [10]. Next, we evaluated a rat peritoneal infection model for chemokine production, influx of inflammatory cells and bacterial clearance in the peritoneal cavity directly after the infection with S.aureus or E.coli at different time points. Finally, we investigated the long-term effect of the conventional lactate-buffered and a recently developed bicarbonate/lactate-buffered PDF on the inflammatory response, i.e. chemokine production, neutrophil recruitment and bacterial clearance upon bacterial challenge in vivo. Both our in vitro and in vivo data show that pre-exposure of the peritoneal membrane to different PDF did not change the migration of functional neutrophils upon challenge.
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Subjects and methods |
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Primary EC. Primary EC were isolated by collagenase treatment as described previously [12] from umbilical cords obtained from the Department of Gynaecology and Obstetrics, Hilversum Hospital, after obtaining informed consent. EC were cultured in M199 medium containing 10% new born calf serum, 10% human serum, 2 mM L-glutamine, 50 U/ml penicillin, 50 µg/ml streptomycin, 1 ng/ml basic fibroblast growth factor and 5 IU/ml heparin on gelatin-coated plates and used in passages 23. Purity was checked by immunofluorescence staining with rhodamine-coupled ULEX type 1 lectin (Vector Laboraties, Burlingame, CA, USA).
In vitro model for the peritoneal membrane. Transwell filters (polycarbonate membrane, 12 mm diameter, 3.0 µm pore size; Costar, Corning Inc., NY, USA) were coated overnight with ECM components (see Results). A transwell was inverted and a collar, which fits exactly to the transwell was put on top as described by Mul et al. [13]. MC were allowed to adhere to the bottom side of a transwell filter in the inverted position at 37°C and 5% CO2. After 4 h the collars were carefully removed and the transwells were placed in an upright position with MC upside-down in 12-well culture plates and subsequently cultured for 9 days. Culture medium was refreshed every other day. ECs (150 000 cells in 500 µl) were subcultured on top side of the fibronectin-coated transwell filter and were cultured for 35 days. In order to form a double-layer we first cultured MC on the bottom side for 4/5 days as described above. Then ECs were seeded on top of the same filter and together cultured for another 4/5 days (Figure 1). The culture medium used for transwell double-layer culture was composed of 50% MC medium and 50% EC medium.
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We included several controls for the confluency and integrity of both monolayers on transwells: without chemoattractant to determine passive migration, leakage of Texas Red-labelled dextrans (100 µg/ml; Molecular Probes; 70 kDa) added to neutrophil solution during transmigration and immunofluorescence on parallel cultures using specific antibodies for each cell type (anti-cytokeratin, Dako and ULEX type 1, Vector Laboratories). All experiments with a passive neutrophil migration >6.5% or dextran leakage of >0.5 µg were excluded from analysis (40% of the experiments). To control for cell polarity, ZO-1 staining was performed using a rabbit anti-human ZO-1 (Zymed Laboratories, Inc., San Francisco, CA, USA).
Cell bound enzyme linked immunosorbent assay
MCs were incubated with 200 U/ml TNF or Dianeal in a 1:4 dilution for 24 h. ICAM-1 and VCAM-1 was evaluated by cell bound enzyme linked immunosorbent assay (ELISA) as described before [6].
Electron microscopy
For transmission electron microscopy, transwells were fixed with 1.5% glutaraldehyde in PBS (pH 7.4) at 4°C for several days. The transwell membranes were excised for post-fixation using 1% osmium tetraoxide at 4°C for 1 h. After dehydration, the transwell membranes were impregnated and embedded in a mixture of epon/araldite followed by polymerization at 56°C. Ultra-thin sections were cut with an ultramicrotome (OMU 3, Leica), stained with uranyl acetate and lead citrate, and visualized with an Electron Microscope (CM100; FEI Company, Electron Optics, Eindhoven, The Netherlands).
In vivo studies
Animals. Male Wistar rats were obtained from Harlan CPB (Zeist, The Netherlands) and were allowed to acclimatize 1 week before the experiments started. Animals were maintained under conventional laboratory conditions and were allowed free access to food and water. The Animal Care Committee of the VU University approved the animal experiments described. At the start of the fluid instillation, rats weighted 250270 g, after 10 weeks PD instillation rats were 440470 g.
Rat infection model. Untreated rats (n = 4/group, per time interval) were i.p. injected with 0.5 ml of a suspension containing 3 x 108 colony forming units (c.f.u.) of S.aureus ATCC 25923 and E.coli EB1 (clinical isolate, kindly provided by Dr F. Namavar, Department of Medical Microbiology, VU University Medical Center). Rats receiving 0.5 ml saline i.p. served as controls (unchallenged group). At different time points after injection (0.5, 1, 2, 6 and 24 h) the animals were killed. The peritoneal cells and bacteria were collected from the peritoneal cavity after i.p. injection of 10 ml Hank's balanced salt solution (HBSS, Gibco BRL). Both the number of peritoneal leukocytes and recovered bacteria were expressed per 10 ml (the injected volume). Bacteria present in the cell-free supernatants and cell pellets were plated in a serial dilution range on LB plates and scored 24 h later independently by two investigators. Total values of both determinations (cell-free bacteria + cell-associated bacteria) were used for analysis.
Peritonitis in a rat peritoneal exposure model. PDF was instilled via a peritoneal catheter connected to a s.c. mini vascular access port (Access Technologies, IL, USA) as described previously [9]. Rats were divided into three groups. The first group (n = 20) received daily 10 ml of heat-sterilized, lactate-buffered PDF with a pH 5.5 and containing 3.86% glucose (Dianeal PD4®; Baxter BV), the second group (n = 20) received heat-sterilized bicarbonate/lactate-buffered PDF with a pH
7.4 containing 3.86% glucose (Physioneal®; Baxter BV, Utrecht, The Netherlands). Sex- and age-matched rats served as controls (n = 7). After 10 weeks all animals, including controls, were challenged (24 h after the last PDF administration for the PDF-treated animals), with S.aureus ATCC 25923 (1 x 109 c.f.u., in 0.5 ml). Four hours later, the peritoneal cells and bacteria were collected and the number of viable bacteria was determined, as described above. To address the bacterial adhesion to peritoneal tissues, portions of omental and mesenteric tissues were dissected, spread on a glass slide and stained with Toluidine Blue (1% Toluidine Blue, 1% Borax) for light-microscopic examination. Throughout the experiment, there was a drop-out of 3545% of the animals due to omental wrapping around the tip of the catheter as we described previously [14], which was not significantly different between both fluid-treated groups. After 10 weeks of fluid instillation, 13 rats of the conventional PDF group and 11 rats of the bicarbonate/lactate-buffered PDF group were challenged, as well as the (seven) untreated animals.
Chemokine determinations
Rat cytokine-inducible neutrophil chemoattractant-1 (CINC-1) (CXCL1) and macrophage inflammatory protein 2 (MIP-2) (CXCL3) concentrations in cell- and bacteria-free supernatants of the peritoneal lavage were determined using an ELISA kit for rat CINC-1 (IBL, Tokyo, Japan); detection limit of 5 pg/ml; for rat MIP-2 (BioSource International, Camarillo, CA); detection limit of 10 pg/ml.
Statistical analysis
Data of in vitro studies are presented by mean±SD. Data of in vivo studies are presented as median with interquartile ranges (Figure 4AC) or in box plots (Figure 4DF), with the median value indicated in the box, which includes 50% of the values, and a 95% confidence interval between the whiskers. MannWhitney U tests were performed to determine statistical differences. P-values < 0.05 were regarded as significant.
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Results |
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It appeared that the inverted MC cultures did not reach confluence at all when fibronectin (10 µg/ml in PBS) was used as coating material (Figure 2A). Actually, increased cell numbers were found on the bottom of the wells, indicating that MC fell off the filter, probably during cell division. Apparently, fibronectin-mediated MC adhesion was too weak to ensure upside-down culture. We therefore used fibronectin together with vitrogen and BSA, which is a very rich and adhesive coating. Under these conditions more cells were present at day 3 of culture and this number increased during culture and reached near confluency at day 8 as shown in Figure 2B. The optimal seeding number of MC per transwell is 250 000 cells/well. Increasing this number did not result in an increase of the number of MC that adhered after 4 h incubation.
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Double-layer culture. Double-layers were cultured with MCs on the lower side and ECs on the top side. We observed that ECs influence MC morphology to some extent. MCs showed a more elongated morphology, however, they remain positive for cytokeratin. The change in morphology is, however, not seen anymore in confluent layers. Transmigration across cell monolayers involves interaction with specialized adhesion molecules at the cell surface. It is important for this model, therefore, to ensure that these cells not only form a monolayer, but also get polarized with a distinct apical and basolateral side of the membrane. As tight junctions are only formed in polarized cells, we confirmed by ZO-1 staining that both MC and EC form a polarized layer on either side of the transwell (Figure 2D and E).
Transmigration of neutrophils
Labelled neutrophils were allowed to migrate from the upper compartment to the lower compartment containing 10 nM fMLP or 10 ng/ml IL-8. Both chemoattractants are important in the peritoneal cavity: the bacterial product fMLP mimics a bacterial infection, while IL-8 is produced by macrophages and MCs upon bacterial infection [15]. For IL-8 we observed a dose-dependent increase in migration (data not shown); however, levels >10 ng/ml are rather unphysiological as higher values are not found in peritoneal effluents of peritonitis patients [16]. Migration was greatly enhanced in single MC, single EC and double-layer cultures towards fMLP or IL-8 (Figure 3A). MC monolayers allowed the highest migration (40% for fMLP and
18% for IL-8) and indicating that MCs are rather easily to pass by neutrophils. EC forms a stricter monolayer as only
20% for fMLP and
11% for IL-8 of the neutrophils were able to migrate. Migration across the double layer was similar to EC alone suggesting that in this model the EC layer is the major determinant for migration capacity. Although we observed no additive barrier effect of MC on neutrophil migration, values for passive migration and leakage of dextrans were always lower in the double layer compared with the single EC layer, indicating a contribution of MCs to the barrier in this model. Next, we tested the effect of glucose, a combination of two GDPs or conventional glucose-containing PDF in the lower compartment on neutrophil migration over the transwell cultures upon IL-8. Since the IL-8 driven migration varies among different isolations of primary cells, we present the data as ratio migration + IL-8/migration-IL-8. The passive migration in these experiments ranged from 3.0 to 6.5%. No significant differences in migration between the test solutions and controls were observed (Figure 3B). As a positive control activation of MCs by TNF
for 24 h resulted in a 2-fold increase in IL-8-induced neutrophil migration (data not shown). Consistent with the migration data, the cell bound ELISA data indicate no change in expression of ICAM-1 and VCAM-1 upon conventional dialysis fluid (Figure 3C), whereas these adhesion molecules were clearly up-regulated after TNF
pre-treatment. This indicates that a higher expression of adhesion molecules leads to higher migration and suggests unchanged MC adhesion molecule expression upon exposure to the test solution.
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Chemokine production. In the rat the neutrophil-activating chemokines appear to be functional homologues of three human growth related oncogene (GRO) proteins (CXCL1, CXCL2, CXCL3) and are termed CINC-1, CINC-2 and CINC-3/MIP-2. Besides the GRO proteins, in humans IL-8 (CXCL-8) functions as neutrophil attractant as well, for which no homologue in the rat have been identified. Injection of S.aureus resulted in a marked increase in both CINC-1 and MIP-2 levels at t = 1 h and t = 2 h and returned to baseline levels after 6 h (Figure 4A).
Peritoneal cell influx and cellular composition. From 2 h after introduction of S.aureus an influx of neutrophils was observed (Figure 4B). The highest number of neutrophils was found 6 h after injection with S.aureus. The total number of peritoneal cells followed the same kinetics as the neutrophil influx (data not shown). As anticipated, the total number of peritoneal cells of the unchallenged group (i.e. injection with PBS) remained unchanged over time (1723 x 106 cells). These cells were mainly macrophages, the percentage neutrophils was not increased (<4%).
Peritoneal clearance of bacteria. Within 2 h after injection with S.aureus 37% of the injected bacteria were detected in the cell-free supernatant and the cell pellet (i.e. either bound or taken up), while after 6 and 24 h infection <1% of bacteria were recovered (Figure 4C). These results indicate bacterial adherence within 30 min after inoculation. Furthermore, clearance of bacteria is strongly correlated to the influx of neutrophils in the peritoneal cavity.
Effects of PDF on bacterial clearance in rat PD model
We next set out an infection experiment in a rat model for peritoneal exposure to PDF. During 10 weeks rats were daily treated with conventional, lactate-buffered PDF or with a newly developed bicarbonate/lactate-buffered PDF. We evaluated the chemokine production, neutrophil influx and bacterial clearance 4 h after challenge with S.aureus. At this time-point the inflammatory reaction had started, but not completely resolved as shown in Figure 4AC. This enables us to observe possible effects of PD treatment in the early phase of the inflammatory response.
Chemokine production. We focused upon MIP-2, one of the human GRO homologues in the rat. MIP-2 levels in PDF-untreated rats correlated well with levels observed 4 h after S.aureus injection in the experiment described above (see Figure 4A). On the contrary, MIP-2 levels were equally, dramatically lower (10-fold) in both PDF-treated groups compared with the control group (P<0.0001) (Figure 4D).
Peritoneal cell influx and cellular composition. Despite an apparent higher number of peritoneal cells recovered from both PDF-treated groups, no significant differences were found among the three groups (median: 92.0 x 106; 77.7 x 106; 57.4 x 106 in the conventional, bicarbonate/lactate and control group, respectively). As expected, recruited cells were predominantly neutrophils (6680%), with no significant differences in the absolute number of neutrophils among the three groups (Figure 4E). The data demonstrate that despite a clearly hampered chemokine response (Figure 4D) the recruitment of peripheral neutrophils towards the peritoneal cavity is not significantly impaired upon peritoneal exposition to PDF.
Peritoneal clearance of bacteria. The total number of recovered bacteria (both cell-associated and in supernatants) showed no significant differences among the groups (Figure 4F), although the median number of bacteria found in the bicarbonate/lactate-buffered PDF-treated group was about half of that in the lactate-buffered PDF-treated group. These results were comparable with the experiment described above (Figure 4C). Light microscopic observation of the omentum revealed that the vast majority of the adherent bacteria were taken up by adherent neutrophils (data not shown), without any obvious differences among the three groups. In addition, just occasionally a few bacteria were found to be cell-free (not taken up by neutrophils) in the omentum. In contrast, no bacteria or neutrophils had adhered to the mesentery.
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Discussion |
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An in vitro model for the peritoneal membrane has to meet a number of criteria: (i) the cells should be as differentiated as possible and (ii) migration should occur in the physiological direction (from apical to basal side of EC, passing ECM and subsequently from basal to apical side of MC); and the model should have the possibility to apply chemoattractants or PDF at the mesothelial side. In our transwell culture system we accomplished this by growing primary human MC on the lower, and primary human EC on top of an ECM-coated porous filter. A model for the peritoneal membrane has been described before, culturing MC on top and EC on the lower side of a filter [17]. However, neutrophil migration in their model was directed upwards, i.e. from lower to upper compartment, which we do not understand, since cells cannot move against the gravity forces without substratum. Before we performed the crucial experiments we had to overcome some difficulties, which include the upside-down culture of the MC, the confluency of both monolayers, the tendency of MC to creep through the pores of the filter, and the variation in response among the various donors of the primary cells. This indicates that the model requires some skills to work with. Comparison of the induced migration over monolayers of EC, MC or the double cell layer clearly show a minor contribution of the MC, compared with EC to the barrier function for neutrophils. Because in these experiments the confluency of the monolayers is carefully controlled, we believe that the junctional complexes between MC are less tight compared with those of the EC. Indeed, in our experience it is barely possible to measure electrical resistance over monolayers of MC, whereas this is easy to achieve with confluent EC. Although we demonstrated an increased ICAM-1 and VCAM-1 expression along with an increased neutrophil migration after stimulation of the MC with TNF-, no changes of both parameters could be found after repeated exposure of the MC with glucose, GDPs or conventional PDF. A limitation of these model studies is the fact that we had to dilute the conventional PDF 1:4 to a final glucose concentration of
55 mM under neutral pH conditions. In higher concentrations MC loose confluency, which make migration experiments not interpretable. This is of course different from the situation in the PD patient, where the mesothelium is constantly exposed to high glucose concentrations at acidic pH. We therefore designed an additional in vivo experimental approach.
Our results clearly show a strongly impaired MIP-2 (rat analogue for GRO) response upon bacterial challenge in the rats chronically exposed to either PDF, without differences between conventional lactate- and bicarbonate/lactate-buffered PDF. Both PDFs have equal, high levels of glucose, which has been demonstrated to be the causative agent in the impaired endotoxin-induced MIP-2 production in alveolar macrophages in diabetic rats [18]. In contrast, infected naive rats display a full-blown chemokine response with levels similar to that found in the previous experiment (Figure 4A). This finding contrasts with some in vitro data showing a better preservation of peritoneal cell functions upon exposure to bicarbonate/lactate solutions compared with lactate-buffered PDF [5,19]. This difference might be explained by the chronic vs short-term approach in the in vivo and in vitro experiments, respectively. Although the chemokine response during episodes of peritonitis has been described [4], this is the first report that shows an inhibitory effect of PD treatment on chemokine response in an in vivo model. Unexpectedly, despite a 10-fold reduction in MIP-2 production, the neutrophil influx in PDF-treated rats was identical compared with naive challenged rats. Several reasons might explain this finding. It could be that the MIP-2 production in PDF-treated rats (although 10-fold lower compared with control rats) is high enough to induce a full-blown neutrophil influx. Besides to that, we inoculated 1 x 109 c.f.u. S.aureus, which is a substantial bacterial load. It thus could be that complement factors like C3a and C5a have been released together with other chemoattractants produced by the bacteria, which could explain an unchanged influx of neutrophils in PDF-treated animals. We cannot exclude that in the case of a bacterial challenge with fewer bacteria, differences in neutrophil recruitment would have been found. The fact that neutrophil migration over the peritoneal membrane in PDF-treated rats is normal points again to the fact that apparently the mesothelium plays a minor role as a barrier for neutrophils, a conclusion we already drew from our in vitro experiments (see above). From earlier studies we know that the mesothelium is strongly injured upon long-term exposure to PDF. This damage is more explicit in the lactate-buffered than in the bicarbonate/lactate-buffered PDF [9]. Apparently, when the neutrophil has passed the endothelial layer of the venules/capillaries, the presence (control rats) or absence (PDF-treated rats) of an intact MC layer seems not really to contribute to the influx of these cells.
Not surprisingly, these recruited neutrophils are perfectly able to clear the i.p. bacteria, since these cells have never been in contact with PDF and thus have normal cellular functions. The latter finding seems to contrast our previous finding, where we showed that the ex vivo bacterial clearing capacity of peritoneal cells from rats exposed to lactate-buffered PDF was highly impaired, whereas peritoneal cells from rats exposed to bicarbonate/lactate PDF were just slightly impaired compared with cells from untreated rats [9]. The observed difference between our previous ex vivo study and the present in vivo study can be explained by the fact that the differentiation of the cells was disparate: in the present study the recruited cells were neutrophils, while in the ex vivo experiment cells were predominantly resident macrophages. Furthermore, our in vivo experiment differs from the clinical PD situation. PD patients have always PDF within their peritoneal cavity. Thus, in the case of peritonitis, the recruited neutrophils become immediately exposed to PDF, which clearly may hamper many functions of these cells. In contrast, in our in vivo model the bacterial challenge was done 24 h after the last PDF instillation to ensure maximum anti-bacterial defense as residual peritoneal fluid decreased this capacity [20].
Upon inspection of both omenta and mesenteries of the challenged rats we barely observed free adhered bacteria. However, on the omenta we observed numerous adherent neutrophils engorged with many phagocytosed bacteria. Interestingly, we did not observe this on the mesenteries in either group. This indicates that the omentum plays an important role in the removal of saturated neutrophils and in the homeostasis of the peritoneum in general.
We thus conclude that despite a strongly reduced chemokine response, the peritoneal recruitment of neutrophils upon appropriate stimulus is not hampered by previous exposure to PDF. Besides to that our data suggest that the mesothelium barely contributes to the barrier function of the peritoneal membrane for neutrophils.
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Acknowledgments |
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Conflict of interest statement. None declared.
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References |
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