Pressure-induced leukocyte margination in lung postcapillary venules

Hideo Ichimura,1,* Kaushik Parthasarathi,1,* Andrew C. Issekutz,3 and Jahar Bhattacharya1,2

1Lung Biology Laboratory, Department of Physiology and Cellular Biophysics, and 2Department of Medicine, College of Physicians and Surgeons, Columbia University, Saint Luke's-Roosevelt Hospital Center, New York, New York; and 3Department of Pediatrics, Microbiology-Immunology and Pathology, Dalhousie University, Halifax, Nova Scotia, Canada

Submitted 25 January 2005 ; accepted in final form 3 May 2005


    ABSTRACT
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Although pressure elevation in lung postcapillary venules increases endothelial P-selectin expression, the extent to which P-selectin causes lung leukocyte margination remains controversial. To address this issue, we optically viewed postcapillary venules of the isolated blood-perfused rat lung by real-time fluorescence imaging. To determine leukocyte margination in single postcapillary venules, we quantified the fluorescence of leukocytes labeled in situ with rhodamine 6G (R6G). Although baseline fluorescence was sparse, a 10-min pressure elevation by 10 cmH2O markedly increased R6G fluorescence. Both stopping blood flow during pressure elevation and eliminating leukocytes from the perfusion blocked the fluorescence increase, affirming that these fluorescence responses were attributable to pressure-induced leukocyte margination. A P-selectin-blocking MAb and the L- and P-selectin blocker fucoidin each inhibited the fluorescence increase, indicating that P-selectin was critical for inducing margination. Time-dependent imaging of blood-borne fluorescent beads revealed reduction of plasma velocity during pressure elevation. After pressure returned to baseline, a similar reduction of plasma velocity, established by manually decreasing the perfusion rate, prolonged margination. Our findings show that in lung postcapillary venules, the decrease in plasma velocity critically determines pressure-induced leukocyte margination.

P-selectin; plasma velocity; pulmonary circulation; pressure-induced; rhodamine 6G


THE ROLE OF HEMODYNAMICS in the development of lung inflammation remains inadequately understood. Here, we address consequences resulting from lung vascular pressure elevation. Although large increases of lung vascular pressures induce serious lung injury secondary to fluid accumulation (1, 32) and vascular remodeling (31, 33), even smaller, more benign pressure increases are capable of inducing vascular signaling. In recent, real-time imaging studies of endothelial cells (ECs) lining lung postcapillary venules, we determined that a modest increase of the vascular pressure induces cytosolic and mitochondrial Ca2+ increases (12, 15). These responses lead to mitochondrial hydrogen peroxide (H2O2) production, which in turn increases expression of the leukocyte adherence receptor P-selectin on the EC surface (12).

Because P-selectin is not extensively expressed on the EC surface under quiescent conditions (20), the increased externalization of EC P-selectin indicates that modest vascular pressure elevation is capable of activating proinflammatory EC responses in lung. However, the extent to which these responses include leukocyte recruitment requires clarification, since some reports discount the role of P-selectin in lung leukocyte margination (11, 21, 26).

We tested the possibility that plasma velocity plays a critical role in P-selectin-leukocyte interactions (16, 18) during vascular pressure elevation. To address this, we subjected lung postcapillary venules to pressure challenges similar to our previous study (12) while we quantified leukocyte margination in the venules through fluorescence determinations. Our findings indicate that pressure elevation causes leukocyte accumulation in postcapillary venules through the combined effects of increased P-selectin expression and decreased plasma velocity.


    MATERIALS AND METHODS
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 MATERIALS AND METHODS
 RESULTS
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Fluorescent dyes and reagents. We used the following fluorescent dyes rhodamine 6G (R6G) and MitoTracker Green (Molecular Probes). Vehicle for dyes and agents was HEPES buffer (150 mmol/l Na+, 5 mmol/l K+, 1.0 mmol/l Ca2+, 1 mmol/l Mg2+, and 20 mmol/l HEPES at pH 7.4) containing 4% dextran (70 kDa, Pharmacia Biotech) and 1% FBS (Gemini BioProducts). Drugs used were rotenone, ebselen (Calbiochem-Novabiochem), H2O2, fucoidin, carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), oligomycin, and N-acetyl-L-cysteine (NAC; Sigma Chemical). Antibodies used were nonblocking mouse anti-rat P-selectin MAb RP-2 and blocking MAb RMP-1, which were supplied by the Department of Microbiology-Immunology, Dalhousie Univ., Halifax, Nova Scotia, Canada, and Alexa Fluor 488-labeled goat anti-mouse IgG (Molecular Probes).

Lung preparation. All studies were approved by the Institutional Animal Care and Use Committee. As we previously reported (15, 28), we pump-perfused lungs (14 ml/min) from anesthetized Sprague-Dawley rats (3.5% halothane inhalation and 35 mg/kg ip pentobarbital sodium) with autologous blood (37°C). Baseline pulmonary artery and left atrial (PLA) and airway pressures were held constant at 10, 5, and 5 cmH2O, respectively. To increase pressure in lung postcapillary venules, we increased PLA by raising the height of the venous outflow. Unless otherwise stated, we delivered agents and dyes in Ringer buffer through a catheter introduced retrogradely in the pulmonary venous system (12, 28).

Imaging protocol. Using our described methods (12, 15), we viewed lung postcapillary venules by means of wide-angle microscopy (AX-70; Olympus America) through an image intensifier (Midnight Sun, Imaging Research) or by laser scanning microscopy (LSM 5 Pascal, Zeiss). These venules form the first vascular generation downstream of septal capillaries. We identified vessel location and vessel margins under bright-field conditions, and we obtained images at a focal plane corresponding to the maximum venular diameter. We quantified venular fluorescence using image analysis software (MCID 5, Imaging Research).

Fluorescence protocols: leukocytes. To stain leukocytes, we added R6G (1 µM) directly to the perfusing blood and commenced imaging after 20 min. In one set of experiments, we stained the leukocytes in vitro by adding R6G to the buffy coat obtained by centrifuging the blood (500 g, 20 min). We then reconstituted the blood perfusion by adding back the stained buffy coat.

P-selectin. As in our previously described methods (12), we infused anti-P-selectin MAb (RP-2, 3.5 µg/ml) for 4 min and then switched the infusion to a fluorescent secondary antibody (Alexa Fluor 488-labeled goat anti-mouse IgG, 10 µg/ml) for 4 min. After a 1-min flush with buffer, we monitored residual fluorescence that indicates EC P-selectin expression (12, 28). To determine the time course of P-selectin expression in response to pressure elevation, we repeated this protocol at different time points after the cessation of pressure challenge.

Plasma velocity. To determine the effects of the venular dilatation resulting from pressure elevation, we determined plasma velocity (Vp) by adding fluorescent beads (1-µm diameter, Fluoresbrite; Polysciences, Warrington, PA) in the blood perfusion. We imaged single venules at an image acquisition rate of 150 ms/image. We calculated Vp as the d/t ratio, where d is the distance traversed by a single bead in the venular midaxis in two successive frames, and t is the transit time that is given by the image acquisition rate. We quantified the venular diameter by digital caliper on bright-field images. To reduce Vp, we decreased pump rate from 14 to 7 ml/min while maintaining perfusion pressures constant as above.

Statistics. Group data are means ± SE. Differences between groups were tested by paired t-test for two groups and by the ANOVA-Newman-Keuls test for more than two groups. Significance was accepted at P < 0.05.


    RESULTS
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 MATERIALS AND METHODS
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Leukocyte margination. By both conventional and confocal imaging, venular fluorescence of R6G was weak at baseline PLA of 5 cmH2O (Fig. 1, A and B), indicating that little leukocyte margination occurred in these venules under quiescent conditions. However, the fluorescence increased markedly during a 10-min PLA elevation to 15 cmH2O (Fig. 1, A and B), denoting increased leukocyte margination.



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Fig. 1. Leukocyte fluorescence in postcapillary venules. Wide-angle (A) and confocal (B) images of single postcapillary venules (white dotted lines show venular margins) are shown at baseline [left atrial pressure (PLA), 5 cmH2O] and after a 10-min pressure elevation (PLA, 15 cmH2O). Arrows indicate sites of clumped fluorescence accumulation. Bars are 20 (A) and 10 µm (B); n = 6. R6G, rhodamine 6G; MTG, Mitotracker Green (2.5 µM).

 
Although we did not quantify leukocyte velocity, we detected that even under baseline conditions most leukocytes flowed rapidly in the vessel midline. However, a subset crawled along the vessel walls at considerably reduced velocity, consistent with previous reports of leukocyte rolling in postcapillary venules (11, 16). During pressure elevation, crawling leukocytes progressively halted at different locations and developed aggregates such that R6G fluorescence increased gradually but in a spatially heterogeneous manner along the venular length (Fig. 2A). Accordingly, R6G fluorescence increased gradually during pressure elevation. By contrast, when we lowered pressure to baseline, the aggregates rapidly dispersed and fluorescence decreased almost immediately (Fig. 2A; P < 0.05). Below, we consider these asymmetric fluorescence transients in relation to Vp.



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Fig. 2. Time course of leukocyte margination. Tracings show time course of R6G fluorescence in single venules exposed to indicated PLA. Traces are for control (A, replicated 6 times), transient blood flow arrest by stopping the perfusion pump (B, dots), Ringer wash (C, arrow), and leukocyte-free perfusion (D). Experiments in B–D were replicated 3 times.

 
Within the period of pressure elevation, the fluorescence increase ceased when we stopped flow, and it commenced when we restarted blood flow (Fig. 2B, dots; P < 0.05). During pressure elevation, a buffer wash into the venule completely removed all fluorescence (Fig. 2C; P < 0.05), indicating that the marginated cells could be easily dislodged. In lungs perfused with blood depleted of leukocytes and platelets, pressure elevation failed to increase R6G fluorescence (Fig. 2D; P < 0.05). Furthermore, we elicited the pressure-induced margination response by adding leukocytes that had been stained with R6G in vitro (not shown). Together, these findings rule out nonspecific mechanisms and affirm that the pressure-induced progressive fluorescence enhancement in the postcapillary venule was attributable to margination of leukocytes arriving with the blood.

P-selectin. In our previous studies, PLA elevation induced P-selectin expression in lung postcapillary venules (12, 15). To determine the present role of P-selectin, we infused the venules with either the P-selectin blocking MAb RMP-1 (19) or with fucoidin, a polysaccharide that inhibits both L- and P-selectin (21, 26). Infusion of RMP-1 and fucoidin each separately inhibited the pressure-induced leukocyte margination response (Fig. 3A), whereas buffer infusion had no effect, indicating that the pressure-induced leukocyte accumulation was P-selectin dependent. We reported recently that the mitochondrial blockers rotenone and FCCP and the antioxidants ebselen and NAC block pressure-induced P-selectin expression in lung venules (12). Here, each of these inhibitors blocked pressure-induced leukocyte accumulation (Fig. 3B), indicating that the leukocyte accumulation resulted from mitochondrial mechanisms activated by the pressure increase.



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Fig. 3. Determinant of leukocyte margination. Bars (means ± SE, n = 3 for each) show changes ({triangleup}) of R6G fluorescence from baseline. A: data are for the indicated P-selectin blocking conditions (RMP-1, 7 mg/ml; fucoidin, 100 µg/ml; *P < 0.05 compared with PLA = 5 cmH2O). B: response to a 10-min pressure elevation in venules treated with vehicle (CT), rotenone (RT, 1 µM), carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FC, 400 nM), ebselen (EB, 100 µM), and N-acetyl-L-cysteine (NA, 10 mM). *P < 0.05 compared with CT. C: immunofluorescence of P-selectin shown at different time points after a period of PLA elevation. Each point is means ± SE for 4 determinations. Line was drawn by nonlinear regression (P < 0.05).

 
To determine the dynamics of P-selectin expression, we maintained cell-free buffer infusion in the microvascular bed. As in our previous studies, in situ immunofluorescence determinations indicated that a 10-min pressure elevation to 15 cmH2O increased P-selectin expression (12). However, P-selectin expression remained high in the immediate poststimulus period, returning to baseline with a half time of 20 ± 5 min (Fig. 3C). These findings indicate that after the 10-min pressure stimulus, the surface expression of EC P-selectin continued for a considerable period.

Vp. PLA elevation increased venular diameter by 25% while concomitantly decreasing Vp by 42% (Table 1). After lowering PLA to baseline, we determined the fluorescence decay, which quantified the rate of leukocyte washout from the venule. We compared decay rates at the unmodified, control Vp and with the pump rate decreased to approximate the low Vp that occurred during pressure elevation (Table 1, low Vp). The Vp reduction caused a sixfold increase in the half time of fluorescence decay (Fig. 4, A and B), indicating inhibition of leukocyte washout from the postcapillary venule. Taking these results together with those in Fig. 3C, we interpret that P-selectin was necessary for leukocyte margination but that the release of marginated leukocytes from the venule was enhanced by increasing Vp.


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Table 1. Effects of pressure elevation in postcapillary venules

 


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Fig. 4. Release of marginated leukocytes. Mean ± SE; n = 3 for each bar. *P < 0.05 compared with control. A and B: decay tracings and half times are shown for venular fluorescence of R6G at PLA = 5 cmH2O immediately following a period of pressure elevation. Vp was either unmodified (control) or reduced by decreasing the pump rate (low). C: changes ({triangleup}) of R6G fluorescence shown at control and reduced (low) Vp settings in venules treated as indicated with H2O2 (100 µM) and P-selectin blocking MAb RMP-1 (7 mg/ml). Vp, plasma velocity.

 
To further explore the role of Vp, we gave 5-min infusions of H2O2 (100 µM), which in our previous study increased P-selectin expression to an extent similar to the present pressure challenge (12). H2O2 caused no leukocyte accumulation at baseline Vp (Fig. 4C, far left bar). Moreover, when we reduced Vp by decreasing the pump rate, there was also no leukocyte accumulation (Fig. 4C, second bar from left). However, on decreasing Vp by 42%, H2O2 induced a robust increase of leukocyte accumulation that could be inhibited by the anti-P-selectin antibody (Fig. 4C). These results reaffirmed the synergistic roles played by P-selectin and Vp in inducing leukocyte accumulation in these venules.


    DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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We show here for the first time that a modest increase of pressure induces leukocyte margination in postcapillary venules of the lung. This was evident in the increase of venular R6G fluorescence. Platelets may have contributed to the response (9, 10). However, we believe that the predominant effect was attributable to leukocytes, since by size criteria the marginating cells were larger than platelets. To rule out nonspecific features of the isolated lung preparation that could contribute to leukocyte margination, we confirmed that leukocyte fluorescence was low under baseline pressure conditions, indicating the absence of intrinsic inflammation in these venules. Furthermore, the pressure-induced leukocyte recruitment was reversible, as indicated by reversal of fluorescence to baseline following pressure reduction. Hence, the margination response resulted specifically from mechanisms induced by pressure elevation.

We addressed the potential concern that venular fluorescence could be contributed by R6G internalized in EC. However, a transient arrest of blood flow within the period of pressure elevation concomitantly arrested the fluorescence increase. No fluorescence increase was evident in lungs perfused with leukocyte-depleted blood, whereas the response could be established by specifically loading leukocytes in vitro with R6G. Moreover, EC fluorescence due to R6G uptake was not affected by pressure elevation. These findings indicate that fluorescence increase resulted specifically from margination of blood-borne leukocytes. Inhibitors of mitochondrial reactive oxygen species production, which we have shown previously to block proinflammatory activation of EC (12), inhibited the margination. Hence, we conclude that mitochondrial mechanisms activated the pressure-induced leukocyte margination.

Mechanisms of margination. We were puzzled by the asymmetric fluorescence responses to increase vs. decrease of pressure. Thus, while the pressure-induced margination occurred gradually, pressure reduction caused almost immediate leukocyte removal. Buffer infusion also readily removed the pressure-marginated leukocytes, indicating that the leukocytes were not strongly adherent to the vascular wall. However, a blocking anti-P-selectin MAb completely inhibited pressure-induced margination, indicating a crucial role for P-selectin in the margination response. We considered that in the postpressure elevation period, pressure reduction to baseline might immediately abolish P-selectin expression, thereby releasing marginated leukocytes and accounting for the loss of venular fluorescence. However, contrary to this expectation, P-selectin expression continued in the poststimulus period, indicating that loss of leukocyte fluorescence in the venule was not attributable to loss of P-selectin expression.

To determine possible hemodynamic mechanisms, we quantified Vp. Although at baseline Vp was consistent with reported data for rat lung (26), pressure elevation decreased Vp concomitantly with increase in venular diameter. At the end of the pressure elevation period, we reduced the blood perfusion rate to match the Vp obtained during pressure elevation. Such a reduction caused a major decrease in the postpressure elevation leukocyte removal from the venule, affirming the inverse relation between plasma flow and margination (16). Hence, we interpret that the critical factor that induced margination during the period of pressure elevation was the decrease in Vp. Conversely, at the end of pressure elevation, returning pressure to baseline reinstated the higher Vp, thereby washing out the marginated cells. Furthermore, under baseline conditions, neither decreasing Vp through perfusion rate reduction nor infusion of the P-selectin inducer H2O2 (12) was sufficient alone to increase R6G fluorescence. However, robust increases of R6G fluorescence resulted following infusion of H2O2 in the presence of low Vp. These findings support the notion that the margination response to both pressure elevation as well as H2O2 resulted from the combined effects of P-selectin expression and low Vp.

Leukocyte-endothelial interactions. Our findings are consistent with previous reports that a subset of leukocytes rolls and slides along the wall of the postcapillary venule (11, 16). Our previous reports indicate that P-selectin expression is negligible in these vessels under nonstressed conditions (12, 15), supporting the view that in lung venules baseline rolling is P-selectin independent (11). However, we show here that once P-selectin expression is induced, rolling leukocytes halt and form aggregates along the vessel wall in a P-selectin-dependent manner. Mechanisms that stabilize leukocytes on the P-selectin-expressing endothelial surface may include expression of leukocyte tethers that attach to P-selectin and enable neutrophils to adhere at low shear (29). Also important is the potential role of platelets. In lungs exposed to proinflammatory stresses, the vascular bed recruits platelets (13, 30). Consequently, the formation of platelet-von Willebrand factor complexes on the endothelial surface could form a matrix that captures leukocytes (2). Further studies are required to address the relevance of these mechanisms to the present data.

P-selectin and lung inflammation. Our findings bear on the controversy on the role of P-selectin in lung leukocyte margination. The existence of P-selectin-dependent margination, which is an established mechanism in systemic vessels, is supported in lung by evidence that anti-P-selectin MAb protects against lung injury induced by cobra venom factor (23) and ischemia-reperfusion (22) and that recombinant P-selectin glycoprotein ligand-1 immunoglobulin fusion protein, which antagonizes both P- and E-selectin, inhibits acid aspiration-induced lung injury (17). Furthermore, P-selectin promotes lymphocyte trafficking in lung (5, 7), and P-selectin knockout mice are protected from ischemia-reperfusion lung injury (24). By contrast, evidence rejecting the role of P-selectin in lung leukocyte margination includes findings that leukocyte rolling in postcapillary venules is P-selectin independent (11), that leukocyte sequestration in rat lung induced by 48 h of hyperoxia is primarily ICAM-1 dependent (26), and that bacteria-induced lung leukocyte migration occurs in P/E-selectin knockout mice (21).

An explanation for these differences in the literature might lie in the considerable heterogeneities of receptor expression and hemodynamics that exist between different vascular segments of the lung. It is known that endothelial P-selectin expression varies among different lung segments (4). For example, septal capillaries express P-selectin poorly. Moreover, our present findings indicate that unspecified differences in lung microvascular blood flow may also account for the reported differences in the literature. We suggest that the extent and sites of leukocyte margination in the lung occurs through complex combinations of the extent of P-selectin expression and the extent to which flow stoppage occurs under disease conditions in specific vascular segments. These mechanisms require better understanding.

Clinical significance. Our findings address the increasing understanding that pressure is proinflammatory in lung. Traditionally, pressure increase is thought to increase fluid filtration passively by causing an imbalance of Starling forces (3). However, several reports indicate that the issue may be more complex. Pressure-induced hyperpermeability responses have been reported for both moderate pressure elevation (27) and for severe elevations that cause capillary stress failure (34). High-pressure pulmonary edema causes lung inflammation as indicated by increased white cells and inflammatory cytokines in the bronchoalveolar lavage (6, 14, 25) and increased tumor necrosis factor-{alpha} in the blood (8). Our findings are consistent with these reports. Importantly, we show that even a modest pressure elevation rapidly induces lung leukocyte margination. Although we did not assess leukocyte activation, it is possible that secondary "hits" activate marginated leukocytes, thereby precipitating the permeability and inflammatory effects of high pressure.

In conclusion, our findings emphasize the importance of considering lung hemodynamics in the understanding of lung inflammatory responses. We show here that the mechanosensitive inflammatory effect of maintained pressure elevation derives from a coupling of mitochondria-induced P-selectin expression with decrease of Vp resulting from vascular dilatation. The impact of Vp was sufficiently important that in the poststimulus period, no margination occurred despite continued expression of P-selectin for a considerable period. We speculate that P-selectin-dependent leukocyte margination in the lung might contribute to inflammatory progression in lung regions subject to low plasma flow by ongoing pathological conditions. For example, left ventricular failure might both increase pressure in postcapillary venules and decrease lung blood flow, thereby setting up conditions for P-selectin-dependent leukocyte margination. The extent to which these mechanisms induce margination in different disease conditions requires further consideration.


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This work was supported by National Heart, Lung, and Blood Institute Grants HL-57556, HL-36024, and HL-64896 (to J. Bhattacharya) and HL-75503 (to K. Parthasarathi).


    FOOTNOTES
 

Address for reprint requests and other correspondence: J. Bhattacharya, St. Luke's-Roosevelt Hospital Center, AJA 509, 1000 10th Ave., New York, NY 10019 (e-mail: jb39{at}columbia.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.

* H. Ichimura and K. Parthasarathi contributed equally to this work. Back


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