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Am J Physiol Heart Circ Physiol 290: H365-H372, 2006. First published September 9, 2005; doi:10.1152/ajpheart.00812.2005
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fMLP-stimulated release of reactive oxygen species from adherent leukocytes increases microvessel permeability

Longkun Zhu and Pingnian He

Department of Physiology and Pharmacology, West Virginia University, Morgantown, West Virginia

Submitted 25 April 2005 ; accepted in final form 17 August 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Our previous study (Am J Physiol Heart Circ Physiol 288: H1331–H1338, 2005) demonstrated that TNF-{alpha} induced significant leukocyte adhesion without causing increases in microvessel permeability, and that formyl-Met-Leu-Phe-OH (fMLP)-stimulated neutrophils in the absence of adhesion increased microvessel permeability via released reactive oxygen species (ROS). The objective of our present study is to investigate the mechanisms that regulate neutrophil respiratory burst and the roles of fMLP-stimulated ROS release from adherent leukocytes in microvessel permeability. A technique that combines single-microvessel perfusion with autologous blood perfusion was employed in venular microvessels of rat mesenteries. Leukocyte adhesion was induced by systemic application of TNF-{alpha}. Microvessel permeability was assessed by measuring hydraulic conductivity (Lp). The 2-h autologous blood perfusion after TNF-{alpha} application increased leukocyte adhesion from 1.2 ± 0.2 to 13.3 ± 1.6 per 100 µm of vessel length without causing increases in Lp. When fMLP (10 µM) was applied to either perfusate (n = 5) or superfusate (n = 8) in the presence of adherent leukocytes, Lp transiently increased to 4.9 ± 0.9 and 4.4 ± 0.3 times the control value, respectively. Application of superoxide dismutase or an iron chelator, deferoxamine mesylate, after fMLP application prevented or attenuated the Lp increase. Chemiluminescence measurements in isolated neutrophils demonstrated that TNF-{alpha} alone did not induce ROS release but that preexposure of neutrophils to TNF-{alpha} in vivo or in vitro potentiated fMLP-stimulated ROS release. These results suggest a priming role of TNF-{alpha} in fMLP-stimulated neutrophil respiratory burst and indicate that the released ROS play a key role in leukocyte-mediated permeability increases during acute inflammation.

formyl-Met-Leu-Phe-OH; hydraulic conductivity; neutrophil priming

POLYMORPHONUCLEAR NEUTROPHILS play a key role in host defenses against invading microorganisms. In response to a variety of stimuli, neutrophils release large quantities of reactive oxygen species (ROS) via respiratory burst, which are essential for killing bacteria but may also be responsible for tissue injury. Therefore, understanding the regulatory mechanisms of neutrophil respiratory burst and the relationship between neutrophil-released ROS and microvessel permeability will be of great clinical significance for the development of therapeutic strategies for inflammatory diseases that preserve the essential functions of neutrophils against bacteria while preventing collateral effect on tissue injury.

Some studies indicate that the physical contact between leukocyte and endothelium is the critical step for endothelial barrier damage, resulting in protein leakage and tissue edema (6, 21), and that the adhesion process was the trigger for the neutrophil respiratory burst (9, 10, 12, 13, 23). However, our previous studies suggested dissociation between leukocyte adhesion and neutrophil respiratory burst. Our study demonstrated that systemic application of TNF-{alpha} induced a significant number of leukocyte adhesions without a measurable increase in either hydraulic conductivity or solute permeability (32), whereas an increase in microvessel permeability was found by perfusing vessels with formyl-Met-Leu-Phe-OH (fMLP)-stimulated neutrophils in the absence of leukocyte adhesion (33). Our present study aims to further investigate the mechanisms that regulate neutrophil respiratory burst and the relationship between ROS released from adherent leukocytes and microvessel permeability in intact microvessels by using combined in vivo and in vitro experimental approaches.

The experiments are designed to investigate whether fMLP can induce respiratory burst from TNF-{alpha}-induced adherent leukocytes resulting in increases in microvessel permeability via ROS release. To accomplish this, a technique that combines single-microvessel perfusion with autologous blood perfusion was employed in venular microvessels of rat mesenteries. Leukocyte adhesion was induced by systemic application of TNF-{alpha}. Microvessel permeability was assessed by measuring hydraulic conductivity (Lp). Changes in microvessel Lp in the presence of TNF-{alpha}-induced adherent leukocytes were investigated before and after the application of fMLP. The mechanisms of the Lp changes associated with fMLP-stimulated adherent leukocytes were explored via the pretreatment of microvessels with ROS scavenger superoxide dismutase (SOD) or an iron chelator, deferoxamine mesylate (DFO). The role of TNF-{alpha} in neutrophil respiratory burst was investigated by measuring chemiluminescence (CL) activity in isolated rat neutrophils.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal preparation. Experiments were carried out in venular microvessels in rat mesenteries. All procedures and animal use were approved by the Animal Care and Use Committee at West Virginia University. Female Sprague-Dawley rats (2–3 mo old, 220–250 g, Hilltop Laboratory Animal, Scottsdale, PA) were anesthetized with pentobarbital sodium given subcutaneously. Female rats were used because the mesenteric vascular bed is more developed than in male rats, making it easier to select a suitable venular microvessel for cannulation, thus requiring the use of fewer rats. The initial dosage was 65 mg/kg body wt with an additional 3 mg/dose given as needed. The trachea was intubated, and a midline surgical incision (1.5–2 cm) was made in the abdominal wall. The mesentery was gently removed from the abdominal cavity and spread over a pillar for measurements of Lp. The upper surface of the mesentery was continuously superfused with mammalian Ringer solution at 37°C. All experiments were carried out in venular microvessels with diameters ranging between 40 and 50 µm with one experiment per animal.

Measurement of Lp. All measurements were based on the modified Landis technique, which measures the volume flux of water across the microvessel wall (8). The assumptions and limitations of the original method and its application in mammalian microvessels have been evaluated in detail elsewhere (8, 18). Briefly, a single venular microvessel was cannulated with a glass micropipette and perfused with albumin-Ringer solution (control) containing hamster red blood cells as markers. A hydrostatic pressure (range 50–80 cmH2O), controlled by a water manometer, was applied through the micropipette to the microvessel lumen. The initial water flow per unit area of microvessel wall [(Jv/S)0, where Jv is water flux and S is unit area of the microvessel wall] was calculated from the velocity of the marker cell after the vessel was occluded, the vessel radius, and the length between the marker cell and the occlusion site. Microvessel Lp was calculated from the Starling equation, Lp = (Jv/S)0/{Delta}P, where {Delta}P is the effective hydrostatic and oncotic pressure difference across the microvessel wall.

Lp measurements before and after TNF-{alpha}-induced leukocyte adhesion. In each experiment, control Lp was measured first with albumin-Ringer perfusate in a selected venular microvessel, which had less than two adherent leukocytes per 100 µm of vessel length. Then the cannulation pipette was removed and blood flow was resumed in that vessel. TNF-{alpha} (3.5 µg/kg body wt) was then injected into the bloodstream through the femoral vein. This amount of TNF-{alpha} applied to each rat was equivalent to ~50 ng/ml of blood based on the estimated blood volume of each rat. The plasma concentration of TNF-{alpha} measured with ELISA was ~500 pg/ml after intravenous injection for 30 min and decreased to 135 pg/ml in 2 h (32). This dosage of TNF-{alpha} was sufficient to induce significant leukocyte adhesion under our experimental conditions, which is also comparable to the plasma TNF-{alpha} concentration in a rat ischemic model (27). Two hours after TNF-{alpha} injection, the vessel in which the control Lp was measured was recannulated and perfused with albumin-Ringer perfusate. The perfusion pressure was kept at 20 to 30 cmH2O to minimize the shear force and retain the adherent leukocytes. Changes in Lp were measured immediately after the recannulation. The wall shear rate calculated based on the Newtonian definition [{gamma} = 8 x (Vmean/D), where Vmean is the mean velocity of marker cells and D is the vessel diameter] was between 28 and 40 s during perfusion. The rolling, tethering and nonfirmly adherent leukocytes were washed away during the initial perfusion period. The adherent leukocytes that could withstand that magnitude of shear rate and remained attached to the vessel wall after the early Lp measurements (which took ~5–10 min) were counted under the microscope and expressed as the number of adherent leukocytes per 100 µm of vessel length.

Measurements of Lp when adherent leukocytes were exposed to fMLP. These experiments were designed to examine whether fMLP can stimulate ROS release from TNF-{alpha}-induced adherent leukocytes, resulting in increases in microvessel permeability. The procedures to induce leukocyte adhesion were the same as stated above. After Lp measurements in the presence of TNF-{alpha}-induced adherent leukocytes, fMLP (10 µM) was applied to either perfusate or superfusate. To maximize the local concentration of ROS released from the adherent leukocytes on fMLP stimulation, perfusate was kept stationary (perfusion pressure set at balance pressure) for 5 min before Lp measurements. To ensure that the 5-min stationary flow does not change Lp, the experimental procedure control was conducted in two of the microvessels in which the same maneuver was applied, with the exception of the application of fMLP.

The effects of SOD and DFO on the Lp changes induced by fMLP-stimulated adherent leukocytes were studied in different groups of microvessels. SOD or DFO was applied after TNF-{alpha} induced leukocyte adhesion and before the fMLP application. The changes in Lp were measured before and after the addition of fMLP to the superfusate in the presence of adherent leukocytes that were pretreated with either SOD or DFO.

Isolation of rat neutrophils. Blood was collected from male Sprague-Dawley rats (2–3 mo old, 350–400 g) by catheterization through the carotid artery and anticoagulated with 3.8% sodium citrate (9:1 vol/vol). A two-step discontinuous Percoll gradient centrifugation method was used to isolate neutrophils from whole blood (33). Male rats were used as blood donors because their body weight is larger than that of female rats at the same age, and their use provides a larger quantity of blood and reduces the number of animals used for the experiments. Our previous study (33) demonstrated no significant difference in neutrophil responses to fMLP between male and female rats.

Measurement of CL. ROS production from TNF-{alpha} and/or fMLP-stimulated neutrophils was quantified by measuring luminol-enhanced cellular CL with the use of a computer-controlled luminometer equipped with reagent injector and thermocontrolled chamber (AutoLumat Plus LB953, Berthold Technologies). Neutrophils were preincubated at 37°C for 10 min before the CL measurement. Each sample (0.25 ml) contained 0.5 x 106 neutrophils with 0.8 µM luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, Sigma). The reaction to fMLP (10 µM final concentration) or TNF-{alpha} (5 ng/ml final concentrations) was initiated by automated injection to ensure an immediate mixing and measurement. For measurements involving an antioxidant agent or an ion chelator, each reagent was added before the incubation. The priming role of TNF-{alpha} in fMLP-stimulated ROS release was examined in isolated neutrophils that were preincubated with TNF-{alpha} in vitro or preexposed to systemically administered TNF-{alpha} in vivo. The CL in each sample was continuously recorded for 10 min and reported as relative light units (RLU per 0.5 x 106 neutrophils). The number of experiments, n, represents the number of animals used in each group of experiments. Duplicate assays were conducted with neutrophils from each rat, except where noted otherwise.

Solutions and reagents. Mammalian Ringer solution was used for dissecting mesenteries, superfusing tissue, and preparing the perfusion solutions. The composition of the mammalian Ringer solution was described previously (33). All perfusates used for control and test perfusion contained BSA (10 mg/ml).

Recombinant rat TNF-{alpha} was purchased from Biosource International (Camarillo, CA). The chemotactic peptide fMLP was purchased from Calbiochem (San Diego, CA). All other reagents were from Sigma. The stock solution of fMLP (10 mM) was prepared with 100% DMSO, and the final concentration (10 µM) was achieved by 1:1,000 dilution with albumin-Ringer solution. All perfusates containing test reagent were freshly prepared before each cannulation.

Data analysis and statistics. In each experiment, if Lp is relatively constant throughout the time course, the mean Lp value for each perfusate was calculated from all of the occlusions during that perfusion period. If a transient increase in Lp is observed, Lp is reported as the means of peak and sustained values. Changes in Lp were expressed as the ratio of testing Lp versus control Lp (Lptest/Lpcontrol). All values in the text are means ± SE, except where noted otherwise. For statistical comparison, the mean values of Lp (control and test) measured in the same vessel were used as paired data. The significance of the differences within or between groups was evaluated by paired t-test, nonparametric Wilcoxon’s signed rank test, or Mann-Whitney’s U-test. A probability value of P < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effect of TNF-{alpha}-induced adherent leukocytes on microvessel Lp. Experiments were carried out in six venular microvessels in rat mesenteries. The mean control Lp of six microvessels was 1.9 ± 0.2 x 10–7 cm·s–1·cmH2O–1. Two hours after TNF-{alpha} injection and resumed blood perfusion in the experimental vessel, the mean number of leukocytes adhered to the microvessel wall increased from the mean baseline level of 1.2 ± 0.2 to 13.3 ± 1.6/100 µm of vessel length (Fig. 1A). However, Lp measured in the presence of such amounts of adherent leukocytes showed no significant changes from its control value. The mean Lp was 2.0 ± 0.2 x 10–7 cm·s–1·cmH2O–1 (Fig. 1B).



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  		Fig. 1. No increases in microvessel hydraulic conductivity (Lp) associated with TNF-{alpha}-induced leukocyte adhesion. Summary results of 6 experiments. A: mean number of adherent leukocytes before and after systemic application of TNF-{alpha} and autologous blood perfusion for 2 h. B: correlative Lp changes measured in the same vessels and experimental conditions as in A. Lptest/Lpcontrol, ratio of testing Lp vs. control Lp.

 
Effect of exposing TNF-{alpha}-induced adherent leukocytes to fMLP on microvessel Lp. Experiments were conducted in 13 microvessels to examine whether fMLP can stimulate ROS release from TNF-{alpha}-induced adherent leukocytes resulting in increases in microvessel Lp. The mean control Lp of 13 vessels was 2.0 ± 0.1 x 10–7 cm·s–1·cmH2O–1. Systemic application of TNF-{alpha} with resumed blood flow increased leukocyte adhesion from a mean basal level of 1.1 ± 0.1 to 13.2 ± 1.1 per 100 µm of vessel length. In five of the microvessels, fMLP was then applied to the perfusate. Lp measured after 5 min stationary flow transiently increased to a mean peak value of 4.9 ± 0.9 times the control value. In the other eight microvessels, fMLP was applied to the superfusate. The mean peak Lp value was 4.4 ± 0.3 times that of the control. The magnitude increases in Lp between these two groups were not significantly different. Figure 2 shows a typical time course of the Lp changes in a single experiment (see Fig. 6 for summary of the results).



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  		Fig. 2. Formyl-Met-Leu-Phe-OH (fMLP)-stimulated adherent leukocytes increase microvessel Lp. Representative experiment shows time course of Lp changes when TNF-{alpha}-induced adherent leukocytes (18 per 100 µm of vessel length) were exposed to fMLP (10 µM) applied to perfusate.

 


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  		Fig. 6. Relationship between number of adherent leukocytes and correlative changes in microvessel Lp under different experimental conditions. Summary results of 6 groups of experiments showing relationship between number of adherent leukocytes (bottom graph) and changes in microvessel Lp (top graph) with and without application of fMLP. Corresponding bars in top and bottom graphs represent data from the same group of experiments. Neither application of fMLP alone (in the absence of significant leukocyte adhesion; n = 6) nor TNF-{alpha}-induced leukocyte adhesion without application of fMLP (n = 7) changed basal Lp. Exposure of adherent leukocytes to fMLP applied to perfusate (n = 5) or superfusate (n = 8) caused a significant increase in Lp, which was attenuated or prevented by pretreatment of the vessel with DFO (n = 5) or SOD (n = 3). *Significant increase from negative control (P < 0.05). {dagger}Significant decrease from positive control (P < 0.05).

 
To examine whether 5 min stationary flow (perfusion at balance pressure) affects Lp, paired experiments were conducted in the same vessel in two of the microvessels. After leukocyte adhesion, Lp measured before and after 5 min stationary flow without fMLP addition showed no significant changes in basal Lp. In contrast, in the same two vessels, Lp transiently increased to 4.6 ± 0.1 times the control value after the adherent leukocytes were exposed to fMLP for 5 min. Figure 3 shows the time course of the Lp changes in one of the experiments.



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  		Fig. 3. Experimental procedure control. Individual experiment shows that neither TNF-{alpha}-induced leukocyte adhesion alone nor stationary flow (at balance perfusion pressure) for 5 min in the presence of adherent leukocytes changed Lp. Transient increase in Lp occurred after adherent leukocytes were exposed to fMLP (10 µM) applied to superfusate.

 
The effect of fMLP on microvessel Lp in the absence of leukocyte adhesion was examined in six microvessels. The mean control Lp was 1.9 ± 0.1 x 10–7 cm·s–1·cmH2O–1. The application of fMLP (10 µM) to the perfusate did not cause a significant change in Lp, which was 1.8 ± 0.2 x 10–7 cm·s–1·cmH2O–1 (see Fig. 6 for summary of the results).

Superoxide scavenger and iron chelator prevented or attenuated the Lp increase induced by fMLP-stimulated adherent leukocytes. To examine whether the released ROS is responsible for the Lp increases when TNF-{alpha}-induced adherent leukocytes were exposed to fMLP, eight experiments were conducted in vessels that were treated with either DFO, the iron chelator, or SOD, the superoxide scavenger, after leukocyte adhesion. The mean control Lp of eight microvessels was 1.9 ± 0.2 x 10–7 cm·s–1·cmH2O–1. Systemic application of TNF-{alpha} and resuming blood flow in the vessel for 2 h increased leukocyte adhesion from 1.4 ± 0.4 to 13.2 ± 1.2 per 100 µm of vessel length. The application of either DFO (1 mM) or SOD (1,500 U/ml) in the presence of adherent leukocytes did not significantly change the basal Lp but significantly attenuated or prevented the Lp increase induced by fMLP-stimulated adherent leukocytes. The mean peak increase in Lp after fMLP application was reduced to 1.9 ± 0.5 (n = 5) and 1.1 ± 0.03 (n = 3) times the control value, respectively. The time course of representative experiment from each group is shown in Figs. 4 and 5, respectively. Figure 6 summarizes the results.



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  		Fig. 4. Prevention of iron-catalyzed hydroxyl radical formation attenuated the Lp increase induced by fMLP-stimulated adherent leukocytes. Individual experiment shows that application of an iron chelator, deferoxamine mesylate (DFO), to both perfusate and superfusate in the presence of adherent leukocytes (12 per 100 µm of vessel length) did not change Lp but significantly attenuated Lp increase when adherent leukocytes were exposed to fMLP.

 


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  		Fig. 5. Superoxide scavenger prevented Lp increase induced by fMLP-stimulated adherent leukocytes. After systemic application of TNF-{alpha}, leukocyte adhesion increased to 12 per 100 µm of vessel length. Application of SOD in the presence of adherent leukocytes did not change Lp but completely abolished Lp increase when adherent leukocytes were exposed to fMLP.

 
Correlation between the magnitude of Lp increase and the number of adherent leukocytes. If the Lp increase in response to fMLP application was attributed to the ROS release from adherent leukocytes, we expect a correlation between the number of the adherent leukocytes and the magnitude of the increases in microvessel Lp. The Lp changes as a function of the number of adherent leukocytes ranging from 9 to 20 per 100 µm of vessel length with and without fMLP stimulation are plotted in Fig. 7 (n = 19). This graph demonstrates a positive linear correlation between the magnitude of Lp increases and the number of adherent leukocytes that were exposed to fMLP. There was no correlation between Lp and TNF-{alpha}-induced leukocyte adhesion in the absence of fMLP stimulation.



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  		Fig. 7. Correlation between magnitude of Lp increases and number of adherent leukocytes in microvessel wall. Summary results of 19 experiments showing changes of Lp as a function of the number of adherent leukocytes with ({bullet}, n = 13) or without ({circ}, n = 6) the application of fMLP.

 
Priming role of TNF-{alpha} in fMLP-stimulated neutrophil CL. ROS production via neutrophil respiratory burst in response to fMLP and/or TNF-{alpha} stimulation was quantified by measuring luminol-enhanced neutrophil CL. Exposure of isolated neutrophils to fMLP transiently increased CL from a mean resting level of 75 ± 7 RLU to a mean peak value of 626 ± 62 RLU (n = 10). The mean peak response occurred at 44 ± 2 s after fMLP injection and then fell quickly toward basal level. Preincubation of neutrophils with DFO (1 mM) or SOD (1,500 U/ml) significantly reduced the fMLP-stimulated CL to 323 ± 22 (n = 3) and 131 ± 19 (n = 3) RLU, respectively. There was no immediate change in CL when neutrophils were exposed to TNF-{alpha} (5 ng/ml; n = 3). Figure 8A shows the time course of changes in CL from four individual representative experiments. Figure 8B summarizes the results.



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  		Fig. 8. Measurements of chemiluminescence (CL) with fMLP or TNF-{alpha}-stimulated neutrophils. A: time course of changes in CL from 4 individual assays. Pretreatments of neutrophils with either DFO (1 mM) or SOD (1,500 U/ml) significantly attenuated or abolished fMLP-stimulated increase in CL. Exposure neutrophils to TNF-{alpha} (5 ng/ml) alone did not change CL activity. B: summary results of 5 groups of experiments. Each datum is mean ± SE of 6 assays and 2 assays per rat neutrophils with the exception of the group of fMLP alone which is mean of 20 assays from 10 rat neutrophils. *Significant increase from negative control (P < 0.05). {dagger}Significant decrease from positive control (P < 0.05). RLU, relative light units.

 
Because exposure of neutrophils to TNF-{alpha} showed no instant changes in CL, the priming role of TNF-{alpha} in fMLP-stimulated ROS release was further examined. Figure 9 shows the concentration and incubation time-dependent priming activities of TNF-{alpha} on fMLP-stimulated CL. TNF-{alpha} at a concentration of 1 ng/ml showed minimum insignificant priming effect. The maximum priming effect of TNF-{alpha} occurred at a concentration of either 5 or 10 ng/ml with 30 or 40 min of incubation time. The mean peak response to fMLP measured with neutrophils that were preincubated with 5 ng/ml TNF-{alpha} for 30 min was 6.6 ± 1.1 times of that in unprimed neutrophils. Figure 9, inset, illustrates the time courses of fMLP-stimulated CL in primed versus unprimed neutrophils.



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  		Fig. 9. In vitro priming effect of TNF-{alpha} on fMLP-stimulated neutrophil CL. Neutrophils were incubated with TNF-{alpha} (1, 5, and 10 ng/ml) for 4 different time periods between 10 and 40 min after fMLP stimulation. Peak CL ratio of TNF-{alpha}-primed neutrophils to unprimed neutrophils is plotted as a function of incubation time. Significant potentiation of fMLP-stimulated CL activity occurred in neutrophils that were preincubated with TNF-{alpha} at concentrations of 5 or 10 ng/ml for 20–40 min. Maximum effect occurred at 30 or 40 min incubation time. Each datum is mean ± SE of 3 assays and 1 assay per rat neutrophils. Inset: individual time course of CL changes in fMLP-stimulated, unprimed versus TNF-{alpha} (5 ng/ml)-primed neutrophils (30 min incubation time). *Significant increase from the control value (P < 0.05).

 
To examine the neutrophil oxidative status under the experimental conditions whereby Lp was measured, CL was further evaluated in neutrophils primed in vivo via systemic application of TNF-{alpha}. After 1 h of TNF-{alpha} application, blood was withdrawn and neutrophils were isolated. The in vivo exposure of neutrophils to TNF-{alpha} showed no significant change in basal CL (mean RLU was 85 ± 6) but significantly potentiated the fMLP-stimulated CL activity. The mean peak value was 1,913 ± 275 RLU, which is 3.1 times the fMLP-stimulated peak CL in unprimed neutrophils (n = 3). Figure 10 illustrates the differences in magnitude and time course of fMLP-stimulated CL between unprimed neutrophils and neutrophils primed in vivo by systemic application of TNF-{alpha}.



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  		Fig. 10. In vivo priming effect of TNF-{alpha} on fMLP-stimulated neutrophil CL. Preexposure of neutrophils to TNF-{alpha} through systemic application in vivo did not change basal CL but potentiated fMLP-stimulated CL by 3.1-fold. Bar graph shows magnitude difference in fMLP-stimulated CL between unprimed (n = 10) and TNF-{alpha}-primed neutrophils through in vivo application (n = 3). Inset: representative individual time course from each group of experiments. *Significant increase from the control value (P < 0.05).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
With the use of combined in vivo and in vitro experimental approaches, this study quantitatively demonstrated the relationship between leukocyte adhesion, ROS release via neutrophil respiratory burst, and the correlative changes in microvessel permeability. Our main new finding is that an additional stimulus, such as the application of fMLP, is necessary to trigger ROS release from TNF-{alpha}-induced adherent leukocytes resulting in increases in microvessel permeability. In addition, our CL measurements in isolated neutrophils demonstrated that TNF-{alpha} alone was not sufficient to induce neutrophil respiratory burst, but preexposure of neutrophils to TNF-{alpha} in vivo or in vitro potentiated fMLP-stimulated ROS release. Taken together, these results demonstrate a priming role of TNF-{alpha} in fMLP-stimulated neutrophil respiratory burst regardless of adhesion status and indicate a key role for released ROS from activated leukocytes in permeability increases during acute inflammation.

Adhesion is the initial reaction of circulating leukocytes to inflammation or tissue injury. For decades, the interaction of leukocytes with endothelial cells has been considered as the critical event leading to tissue and organ dysfunction. Extensive investigations have, therefore, focused on identifying specific ligands and receptors for the adhesion process to prevent tissue injury (6, 20–22). However, whether leukocyte adhesion directly involves the increases in microvessel permeability remains an unresolved issue. There are studies showing that leukocyte adhesion did not occur at exactly the same sites as plasma leakage (3, 4, 16). Our previous study demonstrated that the systemic application of TNF-{alpha} induced significant leukocyte adhesion without causing increases in microvessel permeability in intact microvessels (32). In addition, we demonstrated that perfusing microvessels with fMLP-stimulated neutrophils increased microvessel permeability in the absence of adhesion (33). Similar results were also reported in C5a-stimulated neutrophils in isolated coronary venules (28). On the basis of these findings, we hypothesized that the mechanisms that activate leukocytes resulting in adhesion are independent from those that induce leukocyte respiratory burst and that the released ROS are responsible for the increases in microvessel permeability.

Our permeability studies were consistent with this hypothesis. Our experiments first reaffirmed our previous observation that TNF-{alpha}-induced leukocyte adhesion has no effect on basal microvessel permeability (32). The experiments then demonstrated that TNF-{alpha}-induced adherent leukocytes could be further activated by fMLP, resulting in increases in microvessel permeability via released ROS. Results summarized in Fig. 8 (CL measurements in isolated neutrophils) and Fig. 6 (Lp measurements) show a close correlation between the amount of released ROS and the magnitude of Lp increases when the same stimuli or additional treatments were applied to either isolated neutrophils or adherent leukocytes in intact microvessels. The application of SOD showed the most efficient inhibition in both CL responses and permeability increases, implicating the released superoxide and superoxide-derived oxidant radicals as the main species responsible for the permeability increases. The application of an iron chelator, DFO, showed partial suppression in both CL responses and permeability increases with similar magnitudes. These partial but significant suppressions indicate the involvement of iron-catalyzed hydroxyl radical formation in CL responses and in the increases in microvessel permeability. Although perfusion of SOD or DFO may affect both leukocytes and endothelial cells that form microvessel walls, CL measurements in isolated neutrophils suggest that the inhibitory effect of SOD or DFO on the Lp increases is mainly attributed to the inhibition of ROS production from fMLP-stimulated adherent leukocytes. Additionally, Fig. 7 demonstrates the linear correlation between the number of adherent leukocytes and the magnitude increases in Lp in response to fMLP, which further supports that the fMLP-stimulated ROS release from adherent leukocytes is responsible for the permeability increase. In all in vivo permeability studies, the neutrophil percentage of adherent leukocytes in the vessel walls was not histologically identified. However, on the basis of the functions of leukocytes, neutrophils are the predominant phagocytes recruited to the circulation in response to acute inflammation. Thus a similar situation is expected to occur in adherent leukocytes under our experimental conditions.

Our previous study (32) demonstrated that TNF-{alpha} induced a significant increase in the expression of CD11b/CD18 in both isolated neutrophils and leukocytes in whole blood, which is consistent with its role in leukocyte adhesion in intact microvessels. Our present study further examined the role of TNF-{alpha} in neutrophil respiratory burst. Measurements of CL in TNF-{alpha}-stimulated neutrophils showed no instantaneous CL responses. This result well explains why there were no increases in microvessel permeability with TNF-{alpha}-induced leukocyte adhesion, indicating that the adhesion process alone in the absence of respiratory burst was not sufficient to increase permeability in intact microvessels.

Neutrophil respiratory burst is dependent on NADPH oxidase, a group of plasma membrane-associated enzymes (2). In response to a variety of stimuli, this enzyme generates large quantities of superoxide anion that serves as the starting material for the production of a vast assortment of reactive oxidants. These oxidants are used to kill invading microorganisms but can also cause tissue damage (2, 7). Although this process has long been studied, the mechanisms that regulate neutrophil respiratory burst remain controversial. Some of the studies indicate that the expression of adhesion molecules is the trigger of respiratory burst (9). Others report that cross-linking of integrins is sufficient for full activation of neutrophils in the absence of any additional stimulus (5). There are also studies reporting that ligation of integrins alone is not sufficient for full activation of neutrophils unless another inflammatory stimulus initiates a second activation signal (17). It has been reported in the literature that a full oxidative response of neutrophils can be achieved by preexposure to a priming agent (7, 29–31), defined as one that does not elicit the effector’s function on its own but potentiates the response of neutrophils to another stimulus. Many cytokines and proinflammatory mediators have been reported as effective neutrophil primers, which convert the neutrophils from a nonresponsive to a responsive status (11, 29). Those studies led us to reconsider whether the activation of leukocytes by TNF-{alpha} is completely independent from fMLP-stimulated ROS release. We tested the priming role of TNF-{alpha} in neutrophil respiratory burst under our experimental conditions. The doses and incubation time-dependent CL measurements demonstrated that preexposure of isolated neutrophils to TNF-{alpha} for 30–40 min at concentrations of 5–10 ng/ml potentiated fMLP-stimulated neutrophil CL activity by six- to sevenfold. Similar studies demonstrated that TNF-{alpha}-mediated neutrophil priming was almost completely blocked by anti-TNF monoclonal antibody (29). We further investigated the role of TNF-{alpha} in neutrophil respiratory burst under the same experimental conditions that were applied to Lp measurements. We measured the CL in neutrophils that were isolated from animals in which TNF-{alpha} was systemically applied to the bloodstream. We consider this experimental condition as in vivo priming. In these experiments, the circulating neutrophils were exposed to TNF-{alpha} applied systemically for about one h before blood was withdrawn. Neutrophils were isolated in the absence of TNF-{alpha}. The basal CL measured in these neutrophils was not significantly different from that of unprimed neutrophils, but the in vivo priming potentiated fMLP-stimulated neutrophil CL activity 3–4 times of that in unprimed cells. The priming effect under our experimental conditions was maintained for at least 2–3 h after the priming agent was removed. These results indicate that the plasma TNF-{alpha} concentration under our experimental conditions is sufficient to prime circulating neutrophils. The in vivo priming-induced enhanced neutrophil respiratory burst also well explains the clinical observation that high plasma levels of cytokines and/or circulating endotoxin was associated with the development of adult respiratory distress syndrome (25) or linked to organ failure and mortality (26).

Our results indicate that neither exposure of isolated neutrophils to TNF-{alpha} alone nor TNF-{alpha}-induced leukocyte adhesion in intact microvessels is sufficient to induce leukocyte respiratory burst resulting in permeability increase. However, the actions of TNF-{alpha} on leukocytes are not completely independent from their oxidative responses that mediate increases in microvessel permeability as we originally hypothesized. These new results indicate that TNF-{alpha} plays an important priming role in leukocyte respiratory burst. An additional stimulus, such as fMLP, is necessary to trigger the enhanced oxidative reactions. Even though neutrophil priming has long been recognized, the interaction between the primed leukocytes with endothelial cells in intact microvessels and the roles of priming-induced enhanced oxidative burst in microvessel permeability have not been studied. Our results are the first that demonstrated the differential roles of primed leukocytes with and without the secondary stimulus-induced respiratory burst in the changes in microvessel permeability in intact microvessels.

In this study, we combined our single-microvessel perfusion technique with autologous blood perfusion, which closely mimics the acute inflammatory situation and also retains the capacity of single-vessel perfusion for precise measurements of vascular function under well-controlled experimental conditions. To maximize the ROS concentration in the vessel lumen during fMLP application, the perfusion was maintained stationary for 5 min before the Lp was measured. This approach enables the experimental conditions during Lp measurements to be more relevant to the acute inflammatory conditions. In the presence of blood flow, ROS released from either circulating or adherent blood cells upon activation were accumulative in the bloodstream. However, during Lp measurements, the albumin-Ringer perfusate washes away the inflamed blood and continuously dilutes the released ROS from the adherent leukocytes during fMLP stimulation in that segment of the vessel wall, which may result in an underestimated effect of released ROS on microvessel permeability. The stationary flow for 5 min during fMLP application allows the released ROS to be accumulated for 5 min in the perfusate before the changes in Lp were measured, which compensates the diluting effect via albumin-Ringer perfusion. The initial Lp changes may more accurately reflect the effect of released ROS on microvessel walls. The most important validation of this procedure is that the 5-min stationary flow itself did not change Lp in the absence of fMLP as demonstrated by the paired experiments (sham and test) conducted in the same vessel (Fig. 3).

TNF-{alpha} was also reported to increase vascular permeability independent of neutrophils, mainly in cultured endothelial monolayers or in pulmonary vasculatures in vivo (14, 15). A recent study (14) in pulmonary microvessel endothelial monolayers reported that TNF-{alpha} (50 ng/ml for 4 h) induced NADPH oxidase-mediated increases in permeability to albumin. Our previous study (32) demonstrated that neither TNF-{alpha}-induced leukocyte adhesion nor perfusion of rat venular mesenteric microvessels with TNF-{alpha} (10–100 ng/ml in the absence of blood cells) up to 2 h altered basal Lp or permeability to {alpha}-lactalbumin. The 2-h time frame was selected because those experiments serve as control for leukocyte adhesion studies, which is the time needed to induce significant leukocyte adhesion with systemic application of TNF-{alpha}. Further investigations are needed to determine whether this discrepancy is caused by differences between cell types, species, and TNF-{alpha} exposure time, or differences between studies in vivo and in vitro.

Currently, the mechanisms underlying priming have not been fully understood. It was suggested that increases in receptor number as well as the alteration of receptor function are involved in the upregulation of neutrophil NADPH oxidase resulting in enhanced ROS production (7, 11). The priming process is relatively rapid (5–30 min, depending on the primer) and was previously considered to be irreversible. However, some studies have demonstrated reversible priming in neutrophils that were originally challenged by platelet-activating factor (7, 19). Recently, priming agents have been recognized for an additional function of delaying apoptosis and hence increasing the functional longevity of these cells at the inflamed site (1, 24).

Our study quantitatively demonstrated that the enhanced ROS release resulting from exposure of primed adherent leukocytes to subsequent stimuli is the key element responsible for the increases in microvessel permeability during acute inflammation. Therefore, further understanding of the mechanisms that regulate neutrophil NADPH oxidase is of great importance in modulating the activation status of neutrophils to prevent tissue injury caused by enhanced ROS release during acute inflammation.


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This study was supported by National Heart, Lung, and Blood Institute Grant HL-56237.


   FOOTNOTES
 

Address for reprint requests and other correspondence: P. He, Dept. of Physiology and Pharmacology, School of Medicine, West Virginia Univ., Morgantown, WV 26506-9229 (e-mail: phe{at}hsc.wvu.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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  1. Akgul C, Moulding DA, and Edwards SW. Molecular control of neutrophil apoptosis. FEBS Lett 487: 318–322, 2001.[CrossRef][Web of Science][Medline]
  2. Babior BM NADPH oxidase: an update. Blood 93: 1464–1476, 1999.[Free Full Text]
  3. Baluk P, Bertrand C, Geppetti P, McDonald DM, and Nadel JA. NK1 receptors mediate leukocyte adhesion in neurogenic inflammation in the rat trachea. Am J Physiol Lung Cell Mol Physiol 268: L263–L269, 1995.[Abstract/Free Full Text]
  4. Baluk P, Bolton P, Hirata A, Thurston G, and McDonald DM. Endothelial gaps and adherent leukocytes in allergen-induced early- and late-phase plasma leakage in rat airways. Am J Pathol 152: 1463–1476, 1998.[Abstract]
  5. Berton G, Laudanna C, Sorio C, and Rossi F. Generation of signals activating neutrophil functions by leukocyte integrins: LFA-1 and gp150/95, but not CR3, are able to stimulate the respiratory burst of human neutrophils. J Cell Biol 116: 1007–1017, 1992.[Abstract/Free Full Text]
  6. Carden DL, Smith JK, and Korthuis RJ. Neutrophil-mediated microvascular dysfunction in postischemic canine skeletal muscle. Role of granulocyte adherence. Circ Res 66: 1436–1444, 1990.[Abstract/Free Full Text]
  7. Condliffe AM, Kitchen E, and Chilvers ER. Neutrophil priming: pathophysiological consequences and underlying mechanisms. Clin Sci (Lond) 94: 461–471, 1998.[Medline]
  8. Curry PE, Huxley VH, and Sarelius IH. Techniques in microcirculation: measurement of permeability, pressure and flow. In: Cardiovascular Physiology. Techniques in the Life Sciences. New York: Elsevier, 1983, p. 1–34.
  9. Del Maschio A, Zanetti A, Corada M, Rival Y, Ruco L, Lampugnani MG, and Dejana E. Polymorphonuclear leukocyte adhesion triggers the disorganization of endothelial cell-to-cell adherens junctions. J Cell Biol 135: 497–510, 1996.[Abstract/Free Full Text]
  10. Delano FA, Forrest MJ, and Schmid-Schonbin GW. Attenuation of oxygen free radical formation and tissue injury during experimental inflammation by P-selectin blockade. Microcirculation 4: 349–357, 1997.[Web of Science][Medline]
  11. Edwards SW. Cell signalling by integrins and immunoglobulin receptors in primed neutrophils. Trends Biochem Sci 20: 362–367, 1995.[CrossRef][Web of Science][Medline]
  12. Gaboury JP, Anderson DC, and Kubes P. Molecular mechanisms involved in superoxide-induced leukocyte-endothelial cell interactions in vivo. Am J Physiol Heart Circ Physiol 266: H637–H642, 1994.[Abstract/Free Full Text]
  13. Gaboury JP, Johnston B, Niu XF, and Kubes P. Mechanisms underlying acute mast cell-induced leukocyte rolling and adhesion in vivo. J Immunol 154: 804–813, 1995.[Abstract]
  14. Gertzberg N, Neumann P, Rizzo V, and Johnson A. NAD(P)H oxidase mediates the endothelial barrier dysfunction induced by TNF-{alpha}. Am J Physiol Lung Cell Mol Physiol 286: L37–L48, 2004.[Abstract/Free Full Text]
  15. Horvath CJ, Ferro TJ, Jesmok G, and Malik AB. Recombinant tumor necrosis factor increases pulmonary vascular permeability independent of neutrophils. Proc Natl Acad Sci USA 85: 9219–9223, 1988.[Abstract/Free Full Text]
  16. Hurley JV. Acute inflammation: the effect of concurrent leucocytic emigration and increased vascular permeability on particle retention by the vascular wall. Br J Exp Pathol 45: 627–633, 1964.[Web of Science][Medline]
  17. Jakus Z, Berton G, Ligeti E, Lowell CA, and Mocsai A. Responses of neutrophils to anti-integrin antibodies depends on costimulation through low affinity Fc{gamma}Rs: full activation requires both integrin and nonintegrin signals. J Immunol 173: 2068–2077, 2004.[Abstract/Free Full Text]
  18. Kendall S and Michel CC. The measurement of permeability in single rat venules using the red cell microperfusion technique. Exp Physiol 80: 359–372, 1995.[Abstract]
  19. Kitchen E, Rossi AG, Condliffe AM, Haslett C, and Chilvers ER. Demonstration of reversible priming of human neutrophils using platelet-activating factor. Blood 88: 4330–4337, 1996.[Abstract/Free Full Text]
  20. Kubes P, Grisham MB, Barrowman JA, Gaginella T, and Granger DN. Leukocyte-induced vascular protein leakage in cat mesentery. Am J Physiol Heart Circ Physiol 261: H1872–H1879, 1991.[Abstract/Free Full Text]
  21. Kubes P, Suzuki M, and Granger DN. Modulation of PAF-induced leukocyte adherence and increased microvascular permeability. Am J Physiol Gastrointest Liver Physiol 259: G859–G864, 1990.[Abstract/Free Full Text]
  22. Kubes P, Suzuki M, and Granger DN. Platelet-activating factor-induced microvascular dysfunction: role of adherent leukocytes. Am J Physiol Gastrointest Liver Physiol 258: G158–G163, 1990.[Abstract/Free Full Text]
  23. Moll T, Dejana E, and Vestweber D. In vitro degradation of endothelial catenins by a neutrophil protease. J Cell Biol 140: 403–407, 1998.[Abstract/Free Full Text]
  24. Moulding DA, Quayle JA, Hart CA, and Edwards SW. Mcl-1 expression in human neutrophils: regulation by cytokines and correlation with cell survival. Blood 92: 2495–2502, 1998.[Abstract/Free Full Text]
  25. Parsons PE, Worthen GS, Moore EE, Tate RM, and Henson PM. The association of circulating endotoxin with the development of the adult respiratory distress syndrome. Am Rev Respir Dis 140: 294–301, 1989.[Web of Science][Medline]
  26. Pinsky MR, Vincent JL, Deviere J, Alegre M, Kahn RJ, and Dont E. Serum cytokine levels in human septic shock. Relation to multiple-system organ failure and mortality. Chest 103: 565–575, 1993.[Abstract/Free Full Text]
  27. Takenaka H, Oshiro H, Kim DD, Thompson PN, Seyama A, Hobson RW II, and Duran WN. Microvascular transport is associated with TNF plasma levels and protein synthesis in postischemic muscle. Am J Physiol Heart Circ Physiol 274: H1914–H1919, 1998.[Abstract/Free Full Text]
  28. Tinsley JH, Ustinova EE, Xu W, and Yuan SY. Src-dependent, neutrophil-mediated vascular hyperpermeability and {beta}-catenin modification. Am J Physiol Cell Physiol 283: C1745–C1751, 2002.[Abstract/Free Full Text]
  29. van Leeuwen HJ, Van Der Tol M, Van Strijp JA, Verhoef J, and van Kessel KP. The role of tumour necrosis factor in the kinetics of lipopolysaccharide-mediated neutrophil priming in whole blood. Clin Exp Immunol 140: 65–72, 2005.[CrossRef][Web of Science][Medline]
  30. Vercellotti GM, Yin HQ, Gustafson KS, Nelson RD, and Jacob HS. Platelet-activating factor primes neutrophil responses to agonists: role in promoting neutrophil-mediated endothelial damage. Blood 71: 1100–1107, 1988.[Abstract/Free Full Text]
  31. Watson F, Robinson JJ, and Edwards SW. Neutrophil function in whole blood and after purification: changes in receptor expression, oxidase activity and responsiveness to cytokines. Biosci Rep 12: 123–133, 1992.[CrossRef][Web of Science][Medline]
  32. Zeng M, Zhang H, Lowell C, and He P. Tumor necrosis factor-{alpha}-induced leukocyte adhesion and microvessel permeability. Am J Physiol Heart Circ Physiol 283: H2420–H2430, 2002.[Abstract/Free Full Text]
  33. Zhu L, Castranova V, and He P. fMLP-stimulated neutrophils increase endothelial [Ca2+]i and microvessel permeability in the absence of adhesion: role of reactive oxygen species. Am J Physiol Heart Circ Physiol 288: H1331–H1338, 2005.[Abstract/Free Full Text]



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