Direct Observations In Vivo on the Role of Endothelial Selectins and α4 Integrin in Cytokine-Induced Leukocyte-Endothelium Interactions in the Mouse Aorta
Abstract—The molecular mechanisms underlying leukocyte recruitment in large arteries have been extensively studied using histological techniques on fixed tissues. However, there are no reports that address the dynamics of leukocyte recruitment in large arteries in vivo. We developed an intravital microscopy technique for direct observation of leukocyte-endothelium interactions in the mouse aorta. Circulating leukocytes were labeled intravasally with rhodamine 6G and microscopically visualized within the aorta, allowing direct analysis of leukocyte rolling and adhesion. In untreated vessels, leukocyte-endothelium interactions were virtually absent. However, local pretreatment with cytokines interleukin-1β and tumor necrosis factor-α induced clear-cut leukocyte rolling and adhesion, compatible with normal blood flow and wall shear rate. High shear decreased rolling leukocyte flux and increased leukocyte rolling velocity, thus decreasing the tendency for firm adhesion. Leukocyte rolling was almost abolished by an antibody blocking the function of P-selectin, whereas function-blocking antibodies against E-selectin and the α4-integrin subunit decreased rolling leukocyte flux to 51±34% (mean±SD) and 59±11% of the value before antibody treatment, respectively. In addition, inhibition of E-selectin function, but not of α4 integrin, resulted in increased leukocyte rolling velocity (from 48±32 to 71±32 μm per second). Taken together, we introduce the first model for direct studies of leukocyte-endothelium interactions in a large artery in vivo and demonstrate cytokine-induced shear-sensitive leukocyte rolling that is critically dependent on P-selectin and modulated by E-selectin and α4 integrin.
Adhesive interactions between leukocytes and arterial endothelium precede leukocyte infiltration to the subendothelial space in pathological events such as atherosclerosis and vasculitis.1 2 Characteristics of such interactions have been extensively studied in postcapillary venules in relation to the inflammatory process. The recruitment of leukocytes has in these vessels been shown to require a multistep process including leukocyte rolling on and firm adhesion to the endothelium and subsequent transmigration through the endothelial barrier.3 Similar mechanisms have been postulated to recruit leukocytes to the subendothelial space in atherosclerosis. However, the detailed characteristics of leukocyte-endothelium interactions in large arteries susceptible to development of atherosclerotic lesions have not been subjected to in vivo investigation.
The molecular mechanisms involved in leukocyte rolling along the endothelium in venules involve several cell adhesion molecules (CAMs) on leukocytes and endothelial cells. Rolling interactions are mediated by endothelial P- and E-selectin interacting with their ligands on leukocytes4 5 and also through L-selectin on leukocytes interacting with its endothelial ligand(s).4 6 In addition, endothelial vascular CAM (VCAM)–1, as well as the intestinal homing receptor for lymphocytes, mucosal addressin CAM-1 (MAdCAM-1), has been shown to mediate rolling through interaction with α4 integrins on leukocytes,7 8 mechanisms bypassing the need for selectins in the adhesion cascade.
Because arterial endothelium has been shown to express the same CAMs as those expressed in venules,9 10 11 the molecular basis for adhesive interactions is supposedly similar in both vessel types. However, whereas venular endothelium is sensitive to several stimuli for CAMs to be expressed and for subsequent leukocyte rolling and adhesion to be induced, this is not the case for the arterial vessels so far studied with intravital microscopy, ie, the arterioles.12 Nevertheless, arteriolar rolling can be induced by certain stimuli such as perivascular laser injury13 or treatment with cytokines such as interleukin (IL)–1β and tumor necrosis factor (TNF)–α.14 15 Interestingly, leukocyte rolling in arterioles is also induced by stimuli such as oxidized LDL and cigarette smoke,16 17 known risk factors for development of atherosclerosis in humans. However, several factors are different in large arteries compared with arterioles or venules, some of which are likely to influence leukocyte-endothelium interactions. This calls for studies on leukocyte-endothelium interactions in large arteries in vivo. However, no such studies have been performed, as a result of the lack of an adequate experimental model.
In this study, we used a newly developed intravital microscopic technique for direct observation of leukocyte-endothelium interactions in the mouse aorta in vivo. By stimulating the aorta with IL-1β and TNF-α, leukocyte rolling and adhesion were induced. In this setting, the influence of arterial hemodynamics on leukocyte rolling, and major molecular mechanisms involved in this event, were investigated.
Materials and Methods
Sixty-nine male C57BL/6 mice weighing 20 to 30 g were obtained from B&K (Stockholm, Sweden). The animals were fed a standard diet and water ad libitum. The experiments were approved by the regional ethical committee for animal experimentation.
Four hours before microscopic observation, mice were anesthetized by inhalation of 1% to 2% isoflurane (Forene, Abbott) in 35% O2. The abdomen was opened through a midline incision and the intestines were retracted, giving access to the dorsal peritoneum. Injections of cytokines (0.5 μg TNF-α and 0.125 μg IL-1β in 0.3 mL in PBS) were given in the retroperitoneal space around the aorta inferior to the renal arteries. The abdomen was then closed with a 5-0 suture.
Under isoflurane anesthesia, catheters were placed in the left carotid artery and in the left jugular vein. Through the venular catheter a continuous infusion of bicarbonate-buffered glucose was allowed. The arterial catheter was connected to a pressure transducer, a Grass amplifier, and an infusion of saline. The total rate of fluid infusion was 0.8 mL/hour. Blood pressure and heart rate were continuously monitored. The rectal temperature was kept at 37°C with a heating pad and an infrared heat lamp. The exposed tissue was superfused with a thermostated (37°C) bicarbonate-buffered saline solution equilibrated with 5% CO2 in nitrogen to maintain physiological pH, or covered with a physiologically buffered hyaluronic acid solution (Healon, 14 mg/mL, Pharmacia-Upjohn). All parameters were recorded on computer using Grass Polyview software and stored for later analysis. Serial blood samples (10 μL) were taken through the carotid catheter. Samples were later analyzed for white blood count (WBC) in a Bürker chamber.
The abdomen was reopened in the midline, and the intestines were retracted and kept moist during the experiment. The aorta was carefully exposed and separated from the vena cava for a distance of 2 to 3 mm immediately inferior to the renal arteries. The mouse was placed under the microscope, and an ultrasonic flowprobe connected to a flowmeter (Transonic T-106 flowmeter, 0.7v flowprobe; sample rate, 200 per second) was placed around the artery. Flow could be manipulated by elevating the flowprobe using a micromanipulator causing partial vessel obstruction. Increased flow as compared with baseline level was seen after obstruction was released. Direct intravital microscopic observations were performed on the abdominal aorta 4 to 5 mm downstream of the flowprobe where laminar flow had been reestablished.
Microscopic observations were made using an intravital microscope (Leitz Biomed) with a water-immersion objective (Leitz U-O, 23×). Epi-illumination fluorescence microscopy (Leitz Ploem-o-pac, filter block M2 illuminated by a cooled infrared-filtered lamp [Osram HBO 200W/4]) was started 2 minutes after labeling of circulating leukocytes with an intravenous injection of rhodamine 6G (0.3 mg/mL, 0.67 mg/kg). Images were televised and recorded on videotape using a Panasonic WV-1900 video camera.
Analysis of In Vivo Experiments
In the aorta, rolling and adhering leukocytes were visualized on the anterior half of the vessel facing the objective. Rolling leukocyte flux was determined as the number of leukocytes passing a 150-μm-long reference line perpendicular to blood flow. Leukocyte rolling velocity was measured as the mean velocity of individual rolling leukocytes during a minimum time of 0.5 seconds. Leukocyte rolling distance was defined as the distance a rolling cell covered either before leaving the field of vision or before detaching from the endothelium. Wall shear rate (WSR) in the aorta was calculated from the online flow measurement (q) and the vessel radius (r) measured from the microscopic image according to the formula γw=4q/πr3 . Mean WSR (MWSR) was defined as the average WSR during a specified time of microscopic observation, and maximum WSR (MaxWSR) was calculated as the highest WSR that occurred during the same time period. Peak systolic WSR for every heartbeat was stable during each period of observation, rendering MaxWSR relevant for systolic shear.
Scanning Electron Microscopy
Animals were perfusion-fixed through the left carotid artery with 2.5% glutaraldehyde in 0.1 mol/L cacodylate buffer (20 minutes, 100 mm Hg) with outflow through severed jugular veins. The aorta was excised and stored in fixative overnight. The vessel was then dissected free from perivascular tissue, opened longitudinally, attached en face on glass slides (Superfrost Plus Gold) using superglue, and stored in buffer. The specimens were then dehydrated in increasing concentrations of ethanol and Freon 113 and, finally, through critical point dehydration with CO2. After gold sputter coating, the aortas were examined in a scanning electron microscope (Philips SEM 515).
Antibodies and Reagents
The antibodies used in vivo in this study were monoclonal antibody (mAb) RB40.34 against mouse P-selectin (30 μg per mouse; Pharmingen), mAb 9A9 against mouse E-selectin (30 μg per mouse; a kind gift from B.A. Wolitzky, Hoffman-La Roche Inc), mAb R1-2 against the mouse α4-integrin subunit (150 μg per mouse; Pharmingen), and control mAb R3-34 (60 μg per mouse; Pharmingen). Recombinant human IL-1β and TNF-α were obtained from R&D systems Inc, and rhodamine 6G was from Sigma.
The data represent the mean±SD of measurements obtained in the indicated number of experiments. Statistical analysis was performed using paired t test and Wilcoxon signed rank test for paired samples. Analysis of the influence of hemodynamic parameters was performed using linear regression. Statistical significance was set at P<0.05. In figures, *, **, and *** denote difference from control value by significance of P<0.05, P<0.01, and P<0.001, respectively.
All mice appeared normal and healthy before and during cytokine stimulation. Systemic leukocyte counts were decreased by pretreatment possibly as a result of an inflammatory response in the abdomen after surgery. In addition, stimulation with TNF-α has in itself been shown to decrease systemic leukocyte count.18 Cytokine stimulation may also favor adhesion of mononuclear leukocytes in abdominal microvessels of cytokine-treated animals,8 thereby increasing the percentage of polymorphonuclear cells in peripheral blood as compared with animals treated with PBS. The systemic leukocyte counts are summarized in the Table⇓.
Hemodynamic Parameters in Mice In Vivo
Blood pressure and heart rate were continuously measured in most experiments. Mean arterial blood pressure ranged between 60 and 100 mm Hg. Heart rate ranged between 400 and 750 bpm. Blood flow in the abdominal aorta was monitored online. A typical recording of aortic blood flow with calculated WSR is shown in Figure 1⇓. Normal blood flow in the aorta was 1.86±0.44 mL/min (n=9) with diameters of 510±22 μm. Baseline values of MWSR and MaxWSR were 2710±2047 and 7646±4676 per second, respectively.
Treatment With IL-1β and TNF-α Induces Leukocyte-Endothelium Interactions in the Mouse Aorta In Vivo
Leukocyte rolling and adhesion were virtually absent in untreated arteries, whereas occasional interactions were seen in some aortas after treatment with PBS. On the other hand, clear-cut leukocyte-endothelium interactions were induced by cytokine treatment (Figure 2⇓). This induction was not totally consistent, and only weak responses were seen in the aorta using IL-1β or TNF-α separately (data not shown). A more distinct response was achieved when using both cytokines in combination, suggesting a synergistic effect of these substances as previously shown for leukocyte rolling in arterioles.15 Video images of rolling and adherent leukocytes in the aorta are shown in Figure 3A⇓ through 3F.
The characteristics of cytokine-induced leukocyte rolling changed during the time course of the experiments. Rolling leukocyte flux decreased slowly from the start of intravital microscopy (data not shown). The same was found in control experiments using fluorescence microscopy on cytokine-treated cremaster muscle arterioles prepared as described previously,15 despite stable rolling flux for at least 40 minutes when using transillumination microscopy. Thus, fluorescence microscopy using rhodamine 6G decreases rolling leukocyte flux. In contrast, the distance and velocity of rolling leukocytes in the aorta at constant MWSR were stable for at least 20 minutes of fluorescence microscopy. However, to avoid problems in interpreting the data obtained by intravital microscopy, the time of observation in each experiment was kept below 10 minutes.
Rolling Leukocyte Flux in the Mouse Aorta Is Sensitive to WSR
The influence of WSR on rolling leukocyte flux was measured in the aorta of 5 cytokine-treated mice in 15-second periods at different flow rates. Vessel diameter and blood flow were manipulated by partial vessel obstruction, yielding values of 380 to 540 μm (472±62 μm) and 0.14 to 5.55 mL/min (1.64±1.11), respectively. MWSR ranged between 270 and 8911 per second (2533±1833) and MaxWSR between 1315 and 21 416 per second (7584±4642). The minor stretch caused when obstructing the vessel by elevating the flowprobe did not influence the adhesive properties of the endothelium in control experiments. To adjust for differences in WBC between animals, rolling flux was normalized by dividing flux values with a factor given by WBC/1×106.
The influences of MWSR and MaxWSR on rolling leukocyte flux are shown in Figure 4A⇓ and 4B⇓, respectively. Rolling leukocyte flux decreased with increasing shear at MWSR above ≈2500 per second. Similarly, rolling flux decreased at MaxWSR >12 000 per second. WSR exceeding these values may stress the molecular bonds between leukocytes and endothelium and destabilize interactions. At MWSR and MaxWSR above 6000 and 18 000 per second, respectively, hardly any interactions occurred. This demonstrates a hydrodynamic limit for leukocyte rolling in this in vivo situation.
At MWSR below ≈2000 per second, rolling leukocyte flux showed a tendency to decrease. However, when we examine the rolling leukocyte flux fraction in Figure 4C⇑, it seems that the fraction of leukocytes rolling along the endothelium continues to increase with decreasing shear even at MWSR below ≈2000 per second. This indicates that the molecular bonds between leukocytes and endothelium are functional at low shear, in accordance with leukocyte rolling seen at the low shear levels present in venules,19 and that the decreased flux was caused by decreased delivery of leukocytes to the endothelium at low flow rates. It seems that in the mouse aorta, a peak in rolling leukocyte flux appears at MWSR ≈2000 per second where the relationship between the adhesive capacity of leukocyte-endothelium interactions and the delivery of leukocytes to the endothelium is optimal.
WSR Influences Leukocyte Rolling Velocity
The qualitative appearance of rolling interactions was altered by WSR. At low shear, most rolling leukocytes rolled in a slow and steady fashion, whereas leukocyte rolling at high shear was unstable and characterized by tethering interactions. Transit time of leukocytes was inversely correlated to MWSR resembling a correlation between shear and leukocyte rolling velocity (r=0.626, P<0.001, Figure 4D⇑).
Effect of Antibodies Against Endothelial Selectins and α4 Integrin on Leukocyte Rolling
The molecular events involved in leukocyte rolling were investigated in separate experiments. Leukocyte rolling was observed for 3 minutes before intra-arterial injections of mAbs against specific adhesion molecules followed by a subsequent 3 minutes of observation. WBC and MWSR were stable during experiments. The effect of antibodies on rolling leukocyte flux is shown in Figure 5A⇓. The antibody RB40.34 against mouse P-selectin almost abolished leukocyte rolling (n=7, P<0.001). In animals treated with the E-selectin mAb 9A9, the flux of rolling leukocytes decreased to 51±34% of the value before treatment (n=10, P<0.001), whereas treatment with the antibody R1-2 against the α4-integrin subunit decreased rolling leukocyte flux to 59±11% (n=4, P<0.01). Treatment with control antibody did not alter rolling leukocyte flux (102±70% of value before treatment, n=5; P=0.952).
The effect of antibody treatment on leukocyte rolling was further characterized by analyzing the leukocyte rolling distance of individual rolling leukocytes (Figure 5B⇑). The mean leukocyte rolling distance after treatment with antibodies blocking function of E-selectin and α4 integrin decreased to 80±23% (P<0.05) and 74±10% (P<0.05) of the values before treatment, respectively. Control antibody had no effect (100.8±17%, P=0.917). Furthermore, leukocyte rolling velocity increased from 48±32 to 71±32 μm per second after treatment with the antibody against E-selectin (155±39% of pretreatment value, P<0.001, Figure 5C⇑) but was not altered either by control (97±22%, P=0.798) or by α4-integrin blocking antibody (117±27%, P=0.308). Slow rolling of leukocytes occurred mostly in certain areas of the observed arteries. This slow rolling was attenuated by function inhibition of E-selectin, indicating that the localized slow rolling was E-selectin dependent. In areas of slow rolling, adherent leukocytes were regularly found.
To further clarify the potential of other mechanisms mediating rolling without involvement of P-selectin, observation time after treatment with P-selectin antibody was prolonged up to 10 minutes after antibody was given. In 7 experiments, with a total observation time after antibody treatment of 60 minutes, only 2 leukocytes passed the reference line in the aorta (0.035 cells/min) as compared with the rolling leukocyte flux of 10.9±7.0 cells/min before antibody treatment. Within the total field of vision of 330×430 μm, 3 leukocytes were observed to attach to and roll on the endothelium, whereas 19 adherent leukocytes released from firm adhesion started to roll and subsequently detached. Thus, rolling was dramatically decreased, but not abolished, by treatment with a function-blocking antibody against P-selectin.
Cytokine Stimulation Induces Platelet-Independent Adhesion of Leukocytes in the Aorta
Adherent leukocytes were visualized by intravital microscopy. However, because the distinction between adherent leukocytes and other cells in the arterial wall could sometimes be difficult, and to investigate whether platelets were involved in leukocyte-endothelium interactions, scanning electron microscopy on en face–mounted aortas was performed. In all specimens, the endothelial lining was intact without adherent platelets. In cytokine-treated aortas, leukocytes were seen adhering to the endothelium both in vessels that had been exposed to intravital microscopy and in those that had not. Some leukocytes were deformed in drop shape with the head in the direction of flow giving the impression that they were rolling on endothelium (Reference 2020 , Figure 6⇓). Untreated aortas had no adherent leukocytes. No leukocytes were covered by platelets.
Leukocyte recruitment to the arterial wall is of importance in different physiological and pathophysiological situations. Numerous studies have addressed the molecular events involved in this process using histological techniques. However, no studies have investigated the dynamic events of arterial leukocyte-endothelium interactions in vivo.
In this study, by using direct intravital microscopy on the mouse aorta, we present data on leukocyte-endothelium interactions in a large artery in vivo. The described technique will be an important new tool when studying the rapid dynamics of leukocyte recruitment in arterial pathophysiology such as in atherosclerosis and in certain types of vasculitis. In the present work, we studied IL-1β– and TNF-α–induced leukocyte-endothelium interactions, because both cytokines are expressed in atherosclerosis21 22 23 and also because they have been shown in various models to induce expression of endothelial CAMs that can be found both in early and in late stages of atherogenesis.9 10 11 24 25 Although cytokine stimulation does not resemble any specific pathology, the adhesive mechanisms investigated in this study and their potential in influencing leukocyte rolling in the mouse aorta are likely to be relevant. Future studies in pathological models will also be possible by using the technique described. Using cytokines, combined treatment with both IL-1β and TNF-α proved to be more efficient in inducing leukocyte-endothelium interactions than use of either substance alone, as previously shown in arterioles.15 The mechanism underlying this synergistic effect may involve a multiplicative effect of these cytokines on intracellular signaling systems and could be of importance in upregulation of CAMs on arterial endothelium. Unfortunately, because the aorta was perfused by whole blood in vivo and the leukocytes were intravenously labeled with rhodamine, we could not identify the subtype(s) of the leukocytes rolling along the endothelium. However, techniques that allow this discrimination are currently under development.
By studying aortas in isoflurane-anesthetized mice, we found that virtually no interactions occurred spontaneously, whereas cytokine treatment induced leukocyte rolling and adhesion that were compatible with normal WSR. However, blood flow and shear may be different, and possibly higher, in other situations, such as in the awake and freely moving animal. Nonetheless, WSR influences rolling leukocyte flux and leukocyte rolling velocity, possibly predisposing leukocyte adhesion to sites of low or fluctuating shear such as the coronary arteries, in curvatures or at branch points, areas that are all prone to development of atherosclerotic lesions.26
In the cytokine-treated mouse aorta, P-selectin was found to be critical for leukocyte rolling. E-selectin and α4 integrin appeared to stabilize rolling interactions inasmuch as blocking function of either one of these CAMs decreased rolling leukocyte flux. This indicates that capture of leukocytes to the endothelium is dependent on P-selectin and that E-selectin and α4 integrin play minor roles in this event. However, limited capture and rolling can still occur after function inhibition of P-selectin. Importantly, despite the dominant role for P-selectin in leukocyte rolling, we could not find any evidence for involvement of activated platelets, as suggested by the lack of platelets adherent to any leukocyte in SEM studies on the aorta, including those leukocytes having a shape typically seen in rolling cells.20 In addition, in previous studies in which platelet-leukocyte interactions have been of importance in leukocyte recruitment, clusters of platelets and leukocytes have been seen tumbling and rolling down the vessels of the microcirculation.17 27 In this study, no such clusters were seen.
The finding that an antibody against E-selectin decreases the number of rolling leukocytes in the aorta contrasts with studies in the microcirculation, in which inhibition of E-selectin function increases rolling flux.14 18 The discrepancy between these observations and the ones observed in the aorta could be due to the high shear levels in large arteries stressing the remaining adhesive bonds after antibody treatment, thereby causing leukocytes to detach, whereas shear may not reach these critical levels in microvessels. This is supported by the fact that function inhibition of E-selectin decreased leukocyte rolling distance. A similar function of α4 integrin as of E-selectin in stabilizing leukocyte rolling is plausible. Thus, these data illustrate that rolling leukocyte flux is dependent not only on capture but also on stability of rolling interactions.
Similarly to previous findings in the microcirculation, blocking function of E-selectin in the aorta increased leukocyte rolling velocity, indicating a role for E-selectin in slow leukocyte rolling.18 In addition, E-selectin–mediated slow rolling in areas of the aorta in which adherent cells often were found supports, and extends to large arteries, previous findings of a role for E-selectin and/or slow rolling in leukocyte adhesion.28 29
The importance of endothelial selectins in cytokine-induced arterial leukocyte-endothelium interactions could explain the delayed formation of atherosclerotic lesions in LDL receptor–deficient mice carrying additional mutations in the genes for these adhesion molecules.30 Furthermore, our findings indicate that although the α4 integrin/VCAM-1 pathway has been implicated in leukocyte recruitment in atherogenesis,9 10 11 25 31 the selectins appear to strongly enhance leukocyte recruitment in arteries in vivo, despite expression of VCAM-1 in cytokine-stimulated mouse aortas as detected by immunohistochemistry (E.E. Eriksson et al, unpublished data, 1999). Thus, although VCAM-1 is expressed on arterial endothelium, it may not be sufficient to efficiently recruit leukocytes in large arteries in vivo. Instead, additional contributions of other CAMs are likely to be required.
In summary, we have studied, through direct intravital microscopy for the first time, leukocyte-endothelium interactions in a large artery in vivo. We have demonstrated that arterial shear forces influence leukocyte-endothelium interactions and could predispose leukocyte adhesion to sites of low shear. In addition, we have shown that cytokine-induced leukocyte rolling in the mouse aorta is highly dependent on initial tethering and rolling on P-selectin, whereas E-selectin and α4 integrin stabilize rolling interactions. The findings give novel insights into dynamic leukocyte-endothelium interactions in large arteries and indicate that leukocyte recruitment in these vessels in vivo is strongly dependent on expression and function of the endothelial selectins.
This work was supported by the Wallenberg Foundation, the Swedish Medical Research Council (Grants 4764, 14X-4342, and 04P-10738), the Swedish Heart and Lung Foundation, the Swedish Foundation for Health Care Sciences and Allergy Research, IngaBritt and Arne Lundbergs Foundation, and Karolinska Institutet. We thank Ingeborg May for help with the scanning electron microscopy and the Haephtes Society for continuous support. We also thank B.A. Wolitzky for supplying the E-selectin mAb 9A9.
- Received July 21, 1999.
- Accepted December 9, 1999.
- © 2000 American Heart Association, Inc.
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