EDITORIAL FOCUS
Leukocyte recruitment in the airways: an intravital microscopic study of rat tracheal microcirculation

Lina H. K. Lim1,2, Bruce S. Bochner2, and Elizabeth M. Wagner1

1 Division of Pulmonary and Critical Care Medicine and 2 Division of Clinical Immunology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21224


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Because of its relative inaccessibility, inflammatory cell extravasation within the airway circulation in vivo has been difficult to investigate in real time. A new method has been established using intravital microscopy in the anesthetized rat to visualize leukocytes in superficial postcapillary venules of the trachea. This technique has been validated using local superfusion of lipopolysaccharide (LPS) and N-formyl-methionyl-leucyl-phenylalanine (FMLP). Basal leukocyte rolling velocity (55.4 ± 9.3 µm/s) and adhesion (1.4 ± 0.3 cells/100 µm) were monitored in postcapillary venules (33.9 ± 1.3 µm diameter). At all time points up to 90 min, these parameters were unaltered in control rats (n = 7). In contrast, vessels exposed to 1 µg/ml of LPS (n = 6) exhibited a 57% reduction in leukocyte rolling velocity and an increase in the number of adherent cells (4.7 ± 1 cells/100 µm, P < 0.05). Superfusion with 0.1 µM of FMLP (n = 6) also resulted in a 45% reduction in rolling velocity and an increase in adherent cells (4 ± 0.7 cells/100 µm, P < 0.05). Histological evaluation confirmed local stimulus-induced leukocyte extravasation. These results demonstrate leukocyte recruitment in the airway microvasculature and provide an important new method to study airway inflammation in real time.

adhesion; inflammation; endotoxin; N-formyl-methionyl-leucyl-phenylalanine


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE EVENTS RESPONSIBLE FOR leukocyte transit from circulation into inflamed tissue sites, such as the peritoneal cavity, have been studied extensively in a variety of animal models. Despite the intensity of investigation into inflammatory lung diseases such as asthma and chronic obstructive pulmonary diseases, it has been difficult to directly visualize leukocyte recruitment into the airways in vivo. This is due primarily to the difficulty in experimentally accessing and studying the airway microcirculation. Thus several endpoint analyses, such as bronchoalveolar lavage (38) or lung biopsies (25), and in vitro studies using isolated cells such as pulmonary endothelial cells (31) and airway epithelial cells (30) have been the principal approaches by which leukocyte transit in the airways has been evaluated. As a result, events regulating the initial processes of leukocyte recruitment in the airway microcirculation have not been successfully studied in real time.

The multistep process of leukocyte recruitment, determined by the sequential activation of specific adhesion molecules on both the endothelium and the leukocyte, has been characterized (26). The four major steps in leukocyte transmigration are rolling, activation, firm adhesion, and migration across the endothelium and the basement membrane. Under normal, noninflamed conditions, many leukocytes patrolling the microvasculature come into brief contact with the endothelium and begin rolling, mainly via selectin interactions. Most leukocytes detach from the endothelium and rejoin the circulation, while a few tether to the endothelium. In an inflammatory condition, chemokines produced by endothelial cells and tissue-resident cells are believed to be presented to the slowing leukocyte, facilitating firm adhesion. This is mediated by beta 1- and beta 2-integrins and their immunoglobulin superfamily ligands such as intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) (5). Subsequently, these adherent leukocytes change shape and flatten onto the endothelial surface, leading to transendothelial migration.

Intravital microscopy, a useful and established technique pioneered in the 1800s by Cohnheim (13), enhanced the study of inflammatory processes by providing direct visual observation of living circulatory beds. This technique has been used extensively in the mesentery (28) and the cremaster muscle (23) as well as organ systems such as kidney (41) and liver (18). In addition to the study of blood flow and changes in vascular permeability, intravital microscopy has been used to evaluate leukocyte trafficking (rolling times and velocities, leukocyte cell flux, adhesion, and emigration) after exposure to a variety of inflammatory stimuli. These important initial phases of leukocyte trafficking cannot be quantified using fixed histological sections. Furthermore, this technique has the advantage that cardiovascular, nervous, and immune systems are all intact, thus providing a more physiologically relevant system for studying leukocyte trafficking in vivo. Although intravital microscopic approaches to studying leukocyte recruitment have not been employed successfully in the airways, microscopic examination of the vessels of the adventitial surface of the trachea has focused on changes in arterial and venular diameters during superfusion of several vasoactive substances (14), tracheal blood flow (21), and measurement of microvascular pressure (3). These studies demonstrated that the vessels of the adventitial surface of the trachea are accessible for study, and surgical exposure exerted minimal deleterious effects. Although it is not clear that responses in these vessels are representative of deeper mucosal vessels, they offer the unique opportunity for in vivo assessment of physiological parameters important to airway function.

We have developed a new application of intravital microscopy focusing on the vasculature of the adventitial surface of the trachea. This model allows direct observation of leukocyte trafficking in postcapillary venules of the airways. Here we describe our experience with this application using the two proinflammatory stimuli lipopolysaccharide (LPS) and N-formyl-methionyl-leucyl-phenylalanine (FMLP). The methods established will facilitate future studies of airway inflammation resulting from chemical and mechanical stimuli and provide an approach to study a wide range of leukocyte and endothelial activators within the airways and in disease.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. Male Sprague-Dawley rats (180-220 g) were obtained from Harlan (Indianapolis, IN) and housed in controlled pathogen-free animal facilities. They were maintained on a standard chow pellet diet with tap water ad libitum using a 12-h light/dark cycle. Each treatment group comprised at least six rats. All procedures were approved by the Johns Hopkins Institutional Animal Care and Use Committee.

Surgical procedures. Rats were anesthetized by intraperitoneal injection with 75 mg/kg of pentobarbital sodium (Sigma, St. Louis, MO) in saline. Each animal was intubated with an endotracheal tube (size 14 catheter; Johnson & Johnson, Arlington, TX) inserted past the level of the thyroid gland and was mechanically ventilated (80 breaths/min, 1.5-2 ml tidal vol) with a small rodent ventilator (Harvard 683; Harvard Apparatus, Holliston, MA). Pulmonary inflation pressure was monitored with a pressure transducer attached to a side port of the tracheal cannula. The femoral artery and vein were cannulated with P10 Luer catheters (Becton Dickinson, Sparks, MD) for the measurement of systemic blood pressure and the infusion of additional anesthetic (80 µg/min), respectively. Systemic arterial blood pressure and pulmonary inflation pressure were monitored and recorded on a Grass recorder (Grass model 79D; Grass Instruments, Quincy, MA).

A midline incision was made in the ventral surface of the neck, and the trachea was exposed. The surrounding muscle was held apart with retractors for the exposure of the trachea, and the tethered muscle served as a reservoir for the superfusion buffer around the trachea. Body temperature was kept constant with a heating lamp directed onto the animal for the duration of the experiment. In initial experiments, body temperature was measured using a rectal thermometer and remained unaltered throughout the experiment.

Intravital microscopy. The exposed trachea was superfused with warmed (37°C) control buffer (lactated Ringer solution; Abbott Laboratories, North Chicago, IL) at a rate of 1.5 ml/min. An equilibration period of 10 min was allowed before recording basal parameters. Buffer with and without LPS (1 µg/ml, Escherichia coli serotype A, Sigma) or FMLP (0.1 µM, Sigma) was superfused onto the exposed trachea (1.5 ml/min) at 37°C for up to 90 min. These two inflammatory stimuli were chosen in an attempt to preferentially activate either the endothelium or circulating leukocytes, respectively. In preliminary studies, lower concentrations of both LPS and FMLP were tested. The concentrations selected were based on a reproducible and obvious increase in the number of rolling and adherent cells.

Single venules (25-40 µm diameter, 100 µm length) were selected in each rat. This size range has been shown to be the site of most leukocyte trafficking and migration in the systemic vasculature (26). Venular diameter was measured either on- or offline using digital calipers. The effects of LPS and FMLP on leukocyte trafficking were quantified offline by measuring 1) leukocyte rolling velocity (Vwbc), 2) leukocyte flux (defined as the number of cells passing through these vessels per unit of time), and 3) the number of adherent cells in the 100-µm-length vessel per time point.

Rolling leukocytes were defined as leukocytes that moved at a velocity less than that of erythrocytes in a given vessel. The number of leukocytes (flux) rolling through the vessel per minute was counted manually during offline analysis. The Vwbc of 10-15 leukocytes entering the vessel was determined by measuring the time required for a cell to move 100 µm along the endothelial wall (Vwbc = 100 µm/t). A leukocyte was defined as adherent to venular endothelium if it remained stationary for >30 s. Adherent cells were measured every 15 min and were expressed as the number per 100 µm length of vessel. Red blood cell velocity (Vrbc) was measured using an optical Doppler velocitometer (Microcirculation Research Institute, Texas A&M University, College Station, TX), and mean red blood cell velocity (Vmean) was determined as Vrbc/1.6. Wall shear rate (SR) was calculated by the Newtonian definition SR = 8,000 × Vmean/diameter of vessel (7).

Instrumentation. To observe the microcirculation in the trachea, the preparation was mounted under a reflected light microscope (Olympus, Melville, NY) with a water-immersion objective lens (×40 magnification; working distance: 3.3 mm; depth of field: 1.52 µm) and an eyepiece (×10 magnification). The preparation was transilluminated with a 12-V, 100-W halogen light source. A color camera (Olympus BX50W1) acquired images that were displayed onto a Sony Trinitron color video monitor (PVM-14N5U; Sony Electronics, San Jose, CA) and recorded on a Sony super-VHS videocassette recorder (Sony SVO-9500 MDP) for 4 min every 15 min for offline analysis. A video time-date generator (Horita II TG-50; Horita, Mission Viejo, CA) projected the time, date, and stopwatch function onto the monitor (Fig. 1).


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Fig. 1.   Intravital microscopy setup.

Blood counts. Leukocyte counts were performed on blood taken from the tail vein immediately before the initiation of the tracheal exposure and at the end of each experiment to monitor for systemic effects of the stimulus superfusion. Total leukocytes were counted using an improved Neubauer hemocytometer after a 1:11 dilution in Turk's solution (crystal violet 0.01% wt/vol in 3% acetic acid).

Histology. Leukocyte accumulation around postcapillary venules of the trachea was assessed histologically. After a 90-min superfusion with vehicle, LPS, or FMLP, tracheas were quickly extracted and fixed in 3% formaldehyde at 4°C overnight. Tissues were cleared, embedded in plastic, sectioned longitudinally, and stained using a differential stain (methylene blue/eosin) to show granulocytes and mononuclear cells. Representative sections were made from three animals in each treatment group.

Statistics. Data are reported as means ± SE of n animals per group. Statistical differences were calculated on original values using one-way analysis of variance followed by a Bonferroni posttest for intergroup comparisons. Paired data were analyzed using the paired Student's t-test. A value of P < 0.05 was taken as significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Baseline parameters measured during control conditions. Leukocyte rolling and adhesion were quantified in all rats to determine baseline at unstimulated levels. Leukocytes rolled along the endothelium with relatively high velocities and presented a minimal extent of adhesion. Leukocyte trafficking parameters and vessel hemodynamic properties before the start of treatment are presented in Table 1 (n = 19). For this basal time point, mean Vwbc was 55.4 ± 9.3 µm/s, whereas adhesion was 1.4 ± 0.3 adherent cells/100-µm vessel. The mean baseline SR of the 19 vessels examined was 256 ± 15 s-1, while venules examined in all animals ranged between 25 and 40 µm in diameter and averaged 33.9 ± 1.3 µm in presuperfused vessels. The mean arterial pressure averaged 104 ± 5 mmHg. A representative venule (100 µm length) is shown in Fig. 2 with three adherent leukocytes.

                              
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Table 1.   Basal leukocyte trafficking and hemodynamic parameters



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Fig. 2.   An image captured from the video recording of a vessel exposed to N-formyl-methionyl-leucyl-phenylalanine (FMLP). Images were recorded at set time points after the start of superfusion. This representative venule (100 µm length and 30 µm diameter) was recorded after 30 min of FMLP (0.1 µM) superfusion with 3 adherent leukocytes (L and small arrows). Large arrows pointing to the black dots indicate the sensors of the optical Doppler velocimeter. Fat (F) globules are marked. The total magnification of the image is ×400.

Effect of LPS and FMLP superfusion on Vwbc. Figure 3 shows the time course of changes in Vwbc in the three experimental groups. Exposure to buffer alone did not significantly alter rolling velocity when later time points were compared with the presuperfusion level (P = 0.25). A histogram of the number of rolling cells and the time taken to move 100 µm is presented in Fig. 4. The distribution for all groups is similar before treatment (time 0; 2.0 ± 0.06 s, 192 cells). In addition, rolling time after 90 min of buffer superfusion (2.63 ± 0.15 s; P =0.08) was not changed.


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Fig. 3.   Leukocyte rolling velocities in tracheal venules. Exposed tracheas were superfused with buffer alone (triangle ), lipopolysaccharide (LPS; 1 µg/ml, ), or FMLP (0.1 µM, black-lozenge ) for 90 min. One venule was examined per rat. The data represent means ± SE of n = 6-7 rats per treatment. * P < 0.05 and # P < 0.05 for LPS and FMLP, respectively, when compared with time 0 of the superfusion.



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Fig. 4.   Distribution of velocities of rolling cells. The time taken for a rolling leukocyte to travel 100 µm was measured at 0 (A), 60 (B), and 90 (C) min after superfusion. The data are rolling times of the total number of leukocytes counted in all rats per treatment. The distribution of cells rolling along a 100-µm length of venular endothelium shifted rightward to longer rolling times by the 60-min time point in both LPS- and FMLP-treated rats (P < 0.0001).

When 1 µg/ml of LPS was used to activate the endothelium locally, a time-dependent reduction in Vwbc was observed (Fig. 3), which reached its nadir at 60 min and was sustained for the duration of the experiment. In this group of rats, leukocytes rolled at an average velocity of 54.3 ± 7.4 µm/s before LPS superfusion began, and the velocity was reduced to 23.4 ± 2.5 µm/s at 90 min of superfusion (P < 0.0001). Figure 4 shows that the distribution of cells rolling along a 100-µm length of venular endothelium shifted rightward to longer rolling times by the 60-min time point (4.6 ± 0.26 s; P < 0.0001).

Superfusion of FMLP, like LPS, resulted in a steady decrease in Vwbc with a maximal reduction at 60 min (Fig. 3). The reduction in rolling velocity reached statistical significance at 30 min (40 ± 2.8 µm/s, P = 0.002) compared with the velocity at time 0. FMLP appeared less effective than LPS in increasing leukocyte rolling times along the vascular endothelium (Fig. 4), although at 60 min, the mean rolling time was significantly increased at 3.3 ± 0.2 s, and at 90 min, the mean time was 2.8 ± 0.1 s (both P < 0.0001 from time 0). Thus both LPS and FMLP superfusion resulted in a significant reduction in leukocyte rolling velocities.

Effect of LPS and FMLP on leukocyte flux in tracheal venules. During control conditions before superfusion of test agents, the number of rolling cells did not differ significantly among the three treatment groups and averaged 18.1 ± 2.4 cells/min. Figure 5 shows the time course of leukocyte flux for the three groups. Leukocyte flux was not altered during superfusion with buffer or LPS over the 90-min time course. However, FMLP superfusion resulted in a significant increase in the number of rolling cells at 30 min (52.6 ± 6.5 cells/min, P = 0.007) and 90 min (44.4 ± 8.5 cells/min, P = 0.046).


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Fig. 5.   Leukocyte flux in tracheal venules. The numbers of cells rolling along the endothelium per minute (cell flux) at a particular cross section of the vessel were counted. The data represent means ± SE of n = 5 rats per treatment. # P < 0.05 for FMLP when compared with time 0 of the superfusion.

Effect of LPS and FMLP on leukocyte adhesion in tracheal vessels. Exposure of the trachea to buffer alone did not alter leukocyte adhesion in the vessels observed (Fig. 6). In contrast, the reduction in Vwbc following LPS superfusion was mirrored by a sharp increase in leukocyte adhesion in the postcapillary venules. This increase was maximal and statistically significant 30 min after the start of the superfusion (from 2.0 ± 0.4 to 6.0 ± 0.6 adherent cells/100 µm; P = 0.008) and was sustained throughout the experiment. A similar pattern was observed when FMLP was superfused over the exposed trachea with an increase in leukocyte adhesion from 1.7 ± 0.6 to 7.3 ± 2.0 adherent cells/100 µm (P = 0.006) at 45 min, so both stimuli significantly enhanced leukocyte adhesion.


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Fig. 6.   Leukocyte adhesion in tracheal venules. Leukocytes stationary for >30 s were described as adherent. The data represent means ± SE of n = 6-7 rats per treatment. * P < 0.05 and # P < 0.05 for LPS and FMLP, respectively, when compared with time 0 of the superfusion.

Effect of stimulus superfusion on SR, vessel diameter, and peripheral blood counts. SR measured at various time points before and during the superfusion of the experimental agents are presented in Table 2. In all vessels examined, irrespective of superfusion stimulus, no significant changes in the SR were observed over the 90-min superfusion. In addition, vessel diameters were not altered from control conditions in all treatments examined after 90 min (data not shown). Therefore, the effects seen in leukocyte rolling and adhesion were not a result of changes in SR or vessel diameters.

                              
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Table 2.   Shear rate of vessels at fixed time points of superfusion

To determine the effect of the local stimulus on circulating cell numbers, differential blood counts were obtained immediately before and after every experiment. Leukocyte counts were higher at the end of each experiment (vehicle, LPS, and FMLP), although they reached statistical significance only with FMLP (Table 3).

                              
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Table 3.   Peripheral blood polymorphonuclear cell counts before and after superfusion

Neutrophil infiltration in tracheal tissue after LPS and FMLP superfusion. To evaluate the extent of extravascular accumulation of leukocytes in the superfused tissue, tracheas were extracted after the experiment and fixed for histology. Control tracheas treated with vehicle alone contained few tissue leukocytes (Fig. 7A). In contrast, when the trachea was superfused with LPS (Fig. 7B) or FMLP (Fig. 7C), an inflammatory influx of leukocytes was observed around vessels close to the ventral surface of the trachea. Most of these migrated cells were neutrophils, with few mononuclear cells. Somewhat unexpectedly, after LPS treatment, deeper regions of tissues located closer to the lumen of the trachea also displayed a neutrophilic infiltrate. Thus superfusion of the surface of the trachea with some inflammatory stimuli can cause both superficial and interstitial inflammation.


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Fig. 7.   Histological characterization of leukocyte migration into the trachea. Exposed tracheas were superfused with buffer alone (A), LPS (B; 1 µg/ml), or FMLP (C; 0.1 µM) for 90 min. Tracheas were excised and sectioned longitudinally. Sections were stained with methylene blue/eosin for characterization of neutrophils. Magnification ×40 (inset in C, ×100). Small arrows point to extravasated leukocytes; c, cartilage; v, vessels.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The main objectives of this study were to establish a reproducible system for direct visualization of leukocytes in the rat tracheal microvasculature in real time using intravital microscopy and to quantify hemodynamic parameters, leukocyte trafficking, and recruitment occurring in the airway circulation. The suitability of the model to study leukocyte trafficking was confirmed with two distinct inflammatory stimuli, FMLP and LPS, which have been studied extensively in other systems and shown under similar conditions to activate primarily leukocytes or the endothelium. In doing so, we have shown that leukocyte recruitment in the rat tracheal microcirculation can be visualized in real time. Furthermore, we have documented that LPS or FMLP can produce similar proinflammatory effects to those in other systemic microcirculatory beds, such as the mesentery (16).

Although Ballard and colleagues (3) have used microscopic techniques to measure tracheal arteriolar and venular diameters during superfusion of vasoactive agonists, to our knowledge, inflammation has not been studied in this model. Attempts have been made to address leukocyte recruitment in the pulmonary circulation within the lung by observing leukocyte rolling and adhesion through a specially designed transparent thoracic window inserted into the chest wall (27). The authors examined fluorescent leukocytes rolling in serosal vessels of the lung and observed that most leukocytes were trapped in the capillary network. The model described in the present study focused on leukocyte kinetics in the vasculature of the adventitial surface of the trachea, which has not been previously characterized in any species. These systemic vessels that course between the cartilaginous rings are supplied by the thyroid arteries. Because leukocyte rolling and adhesion occurred in postcapillary venules in the systemic tracheal circulation, this observation demonstrates a significant difference between the site of leukocyte trafficking in the pulmonary vasculature compared with the systemic vasculature of the lung. Advantages of the technique are that it is relatively noninvasive, requires only minor surgical intervention, and can be used to address the mechanisms of inflammatory cell recruitment to the airways.

Others have used intravital microscopy to study nonairway microcirculatory beds, such as the mesentery, and extrapolated the results to the airways (10). However, it cannot be assumed that leukocyte behavior in the airways is necessarily similar to other microvascular beds. However, the mechanisms of leukocyte recruitment in the tracheal circulation have yet to be determined. The importance of the tracheal circulation in airway inflammation has been reported in several studies. Histological assessment of airway inflammation has focused on endothelial cell adhesion molecule expression (35), vascular permeability (33, 34), angiogenesis (8), and leukocyte accumulation (9) in the trachea. In particular, the pharmacology of neutrophil adhesion and infiltration in rat tracheal venules after several inflammatory stimuli, such as cigarette smoke (4) and allergen challenge (5), has been studied in detail. Inhalation exposure to dry air (6) and ozone (20) also causes inflammatory cell influx into tracheal and lung tissues, demonstrating the relevance of the trachea in respiratory and airway inflammation.

The visual resolution of leukocytes in the tracheal microcirculation is not as high as that of the mesenteric circulation; however, unstained leukocytes can be seen clearly (Fig. 2). Both in vitro and in vivo leukocytes have been stained with a variety of fluorescent dyes to enhance their visualization (11). However, such staining procedures can cause leukocyte activation (1) and thereby alter leukocyte rolling and adhesion kinetics. Thus we chose not to stain the leukocytes and to visualize them in a more physiological state. In this case, individual subpopulations of leukocytes could not be distinguished. However, based on the time course of the leukocyte infiltration into a tissue, the cell type visualized can be predicted and subsequently confirmed by histology.

The parameters used in this study (rolling, flux, and adhesion) have been validated in previous reports (16, 23, 26, 32). In all applications of intravital microscopy, the entire three-dimensional cylindrical vessel (30-40 µm in diameter) cannot be observed simultaneously. In the present study, a cross-sectional plane in the midpoint of the vessel was selected where the majority of inflammatory cells could be visualized. In any case, the number of cells visualized and counted was an underestimation of the total number in real time. Rolling was assessed by measuring the time taken for a leukocyte to move 100 µm along the vessel endothelium. At baseline, leukocytes displayed rolling velocities in the range of 35-50 µm/s, similar to Vwbc found in the rat mesentery (17). However, cell flux, defined as the number of cells passing through these vessels per unit of time, was found to be one-half of that measured in the mesentery (16). This difference may relate to the size of the microcirculation studied. Adhesion was measured as the number of stationary cells motionless for >30 s in the 100-µm vessel. Because many cells start to tether and adhere to the endothelium and subsequently disengage and return into the bloodstream, it is important to measure firm adhesion as defined by the time the cell remained stationary. Firmly adherent cells can also be distinguished as flattened, shape-changed cells, and in control rats, only 1-2 cells were found adherent per 100-µm vessel. This is similar to values found in other rat intravital microscopy studies in other vascular beds (16). Unfortunately, transmigration was not quantified in the present study because this process was not visible due to the thickness of the trachea. This is a limitation of the current model because, when examining thinner tissues in other applications, one can observe extravascular leukocyte accumulation directly (16). However, this problem was partially overcome by performing histology of the tracheas, albeit without the real time component. As expected, most migrated leukocytes were neutrophils.

Control tracheas were surgically exposed and superfused with buffer only. Exposure of the trachea for an extended period of time might induce rolling due to mild endothelial activation. However, it is not possible to predict a true control in unexposed tissues for measurements of leukocyte kinetics. Thus these minimally increased values served, in this case, as "baseline" control.

To validate the model, the effects of two inflammatory stimuli, LPS and FMLP, on leukocyte recruitment were examined. Both local (16) and systemic (37) actions of LPS are well described in other intravital microscopic studies. Superfusion of the exposed trachea with LPS caused tissue inflammation and cellular influx, both superficially as well as in deeper vessels closer to the airway lumen. A time-dependent reduction in Vwbc was observed, mirrored by an increase in leukocyte adhesion over the 90-min observation period. LPS also significantly increased the rolling times of leukocytes along the vessel lumen (Fig. 4), similar to that which was previously shown in the rat mesentery (16). Likewise, exposure of the cat mesentery to LPS induced a steady decrease in Vwbc while increasing leukocyte adherence and emigration, and both SR and vessel diameters were substantially reduced (19). However, when the external surface of the trachea was to exposed LPS, neither SR nor vessel diameter changed compared with control, suggesting that the recruitment caused by LPS in our study was not associated with a reduction in blood flow. Systemically administered LPS induces a mild neutropenia, followed by a marked neutrophilia, due to the release of tumor necrosis factor (TNF)-alpha and subsequent release of neutrophils from the bone marrow (40). In our study, topical application of LPS onto the trachea did not alter the systemic inflammatory cell profile significantly or the overall leukocyte flux. Perhaps a longer superfusion would have induced a systemic neutrophilia, because at 90 min, the values were approaching statistical significance. However, at this time point, the lack of neutrophilia confirms that superfusion of LPS onto the surface of the trachea produced a local inflammatory cell recruitment response.

Further validation of the model was achieved by investigating the effects of FMLP on leukocyte trafficking in the rat tracheal circulation. A bacterial-derived oligopeptide and potent leukocyte chemoattractant, FMLP has been used previously in intravital microscopy studies to induce leukocyte rolling and adhesion in other microcirculatory sites such as the rat mesentery (29) and the hamster cheek pouch (32). Furthermore, with FMLP as a stimulus, leukocyte rolling was found to be critical for leukocyte adhesion (29). In the present study, superfusion of FMLP also enhanced leukocyte adhesion and accumulation in the tracheal tissues. Similar results have been described in the hamster cheek pouch, where adhesion and emigration were increased after FMLP superfusion (32). Interestingly, FMLP was less potent than LPS in increasing leukocyte rolling times but was equally effective as LPS at inducing leukocyte adhesion (Figs. 4 and 6, respectively). This suggests that FMLP does not activate the endothelium but acts directly on leukocytes to induce adhesion as a chemoattractant. These data indicate that FMLP-induced leukocyte activation and adhesion can occur independently of endothelial activation. Unlike LPS, superfusion with FMLP also increased cell flux (Fig. 5), a parameter influenced by SR, vessel diameter, and systemic leukocytosis. Our results showed no changes in SR or vessel diameter; however, peripheral blood neutrophils were increased significantly. The reason for the FMLP-induced neutrophilia is unknown but may relate to its small size and easier accessibility to the circulation.

Although the mechanisms responsible for LPS and FMLP-induced leukocyte extravasation have not yet been explored in this model, specific receptors for each stimulus have been previously described. LPS-induced leukocyte recruitment is related to the sequential activation of LPS-binding protein (36), CD14 (2), and toll-like receptors such as TLR-4 (15). Furthermore, LPS could be activating nuclear factor-kappa B through TLR-4 on the endothelium to regulate responsive genes, inducing the production of proinflammatory cytokines such as TNF-alpha , interleukin (IL)-1beta , and in the rat, cytokine-induced neutrophil chemoattractant, a murine homolog of IL-8 (16). In addition, LPS likely increases the production of chemokines such as RANTES and monocyte chemoattractant protein-1 (24) as well as the expression of E-selectin, ICAM-1, and VCAM-1 on the endothelium (22). In the case of FMLP, previous studies have determined that FMLP binds to specific seven-transmembrane, G protein-linked receptors on granulocytes but not the endothelium (39), triggering intracellular signaling cascades that lead to chemotaxis, superoxide generation, and release of granular products. Furthermore, stimulation with FMLP can also lead to increases in the expression of cytokines such as IL-8 (12) and both expression and function of beta 2-integrins to promote leukocyte adhesion and migration.

We have defined leukocyte kinetics in the vasculature of the adventitial surface of the trachea after superfusion of two inflammatory stimuli. Whether the responses observed in these vessels are representative of deeper, mucosal vessels is not known. Future modifications of the preparation involving surgical sectioning of the trachea above the site of tracheostomy will allow direct visualization of mucosal vessels and confirmation of these initial results.

In conclusion, we have successfully adapted the technique of intravital microscopy to directly visualize leukocyte accumulation in the vasculature of the adventitial surface of the trachea of the rat after local exposure to inflammatory stimuli. Furthermore, we have shown that the technique is sufficiently sensitive to measure changes in hemodynamic parameters as well as leukocyte rolling and adhesion in the tracheal circulation. This novel application of intravital microscopy will allow a thorough examination of the various mechanisms involved in airway inflammation by allowing visualization of leukocyte recruitment in the tracheobronchial circulation. Future studies investigating the effects of mechanical stress and chemical stimuli in the airways, while addressing leukocyte recruitment in the tracheal microvasculature, will provide new insights into the role of the airway circulation in inflammation.


    ACKNOWLEDGEMENTS

We thank Lisa Kostura for histological assessment of tracheas.


    FOOTNOTES

This study was supported in part by National Heart, Lung, and Blood Institute Grant HL-10342.

Address for reprint requests and other correspondence: E. M. Wagner, Johns Hopkins Asthma and Allergy Center, Division of Pulmonary and Critical Care Medicine, 5501 Hopkins Bayview Cir., Baltimore, MD 21224 (E-mail: wagnerem{at}jhmi.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.

First published November 30, 2001;10.1152/ajplung.00261.2001

Received 16 July 2001; accepted in final form 7 November 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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