This study evaluated the changes in the biomechanical properties of endothelial cells (ECs) induced by neutrophil adhesion and the roles of ICAM-1 and reactive oxygen species (ROS) in modulating these changes. Neutrophil adherence to 24-h TNF-α-activated pulmonary microvascular ECs induced an increase in the apparent stiffness of ECs within 2 min, measured with magnetic twisting cytometry. An anti-ICAM-1 Ab blocked the EC stiffening response without inhibiting neutrophil adherence. Moreover, cross-linking ICAM-1 mimicked the stiffening response induced by neutrophils. The neutrophil-induced increase in the apparent stiffness of ECs was inhibited with 1% DMSO (a hydroxyl radical scavenger), allopurinol (a xanthine oxidase inhibitor), or deferoxamine (an iron chelator), suggesting that ROS may be involved in mediating the EC stiffening response. The cellular sources of ROS were determined by measuring the oxidation of dichlorofluorescein. Neutrophil adherence to TNF-α-activated ECs induced ROS production only in ECs, and not in neutrophils. This ROS production in ECs was completely prevented by the anti-ICAM-1 Ab and partially inhibited by allopurinol. These results suggest that ICAM-1-mediated signaling events during neutrophil adherence may activate xanthine oxidase, which in turn mediates the ROS production in ECs that leads to stiffening. ROS generated in ECs on neutrophil adherence appear to mediate cytoskeletal remodeling, which may modulate subsequent inflammatory responses.

Neutrophil sequestration and emigration are important features of inflammatory processes. Neutrophil-endothelial cell (EC)4 adhesion is usually a prerequisite for neutrophil emigration and is often mediated by the neutrophil β2 integrins and EC ICAM-1. Whereas optimal binding of the β2 integrins to their ligands requires conformational changes, ICAM-1 up-regulation is regulated at the transcriptional level by inflammatory cytokines (1, 2).

Recent studies have provided considerable evidence that not only does neutrophil-EC adherence provide physical interactions between these two cell types but it also results in intracellular signaling in both neutrophils and ECs that may modulate neutrophil emigration. For instance, chemoattractant-induced neutrophil adherence and emigration induce intracellular Ca2+ increases (3), F-actin stress fiber formation (4), myosin light chain kinase activation (4, 5, 6), and isometric tension generation (6) in ECs. Inhibition of these changes in ECs reduces neutrophil emigration across the EC monolayer in vitro (3, 4, 5). Moreover, neutrophil adherence to IL-1-activated ECs leads to intracellular Ca2+ increases and F-actin stress fiber formation in ECs (7). Adhesion also induces signaling in neutrophils through integrins, because neutrophil adherence to surface-bound β2 or β3 integrin ligands, including anti-β2 or anti-β3 integrin Abs, ICAM-1, or extracellular matrix proteins, results in reorganization of the actin-based cytoskeleton and ROS production (8, 9, 10).

This study evaluated the effect of neutrophil adherence to human pulmonary microvascular ECs on the functions of these ECs, particularly their biomechanical properties. These properties were measured using magnetic twisting cytometry (11, 12). This technique measures the angular rotation (strain) of ferromagnetic beads bound to cells upon application of a magnetic torque (stress), and the apparent stiffness of the cells is defined as the ratio of stress to angular strain. The roles of ICAM-1, reactive oxygen species (ROS), and nitric oxide, in neutrophil-mediated EC stiffening were examined. Finally, the contribution of ICAM-1-mediated adhesion to the production of ROS within ECs was evaluated. These studies showed that neutrophil adhesion to ECs resulted in production of ROS in ECs that was ICAM-1-dependent and that these ROS were required for neutrophil-induced increase in the apparent stiffness of ECs. This pathway may contribute to the regulation of neutrophil emigration and EC permeability.

Allopurinol, N-methyl-l-arginine (l-NMA), fMLP and deferoxamine were obtained from Sigma (St. Louis, MO); 2′,7′-dichlorofluorescein diacetate (DCFDA) was obtained from Molecular Probes (Eugene, OR); recombinant human TNF-α was obtained from R&D Systems (Minneapolis, MN); murine anti-human ICAM-1 Ab (clone RR1/1) used as a blocking Ab was obtained from Biosource International (Camarillo, CA); murine anti-human ICAM-1 Ab (clone 6.5B5) used for cross-linking and ELISA studies and rabbit anti-mouse IgG Ab were obtained from Dako (Carpinteria, CA); murine anti-human ICAM-1 Ab (clone LB2) was obtained from Becton Dickinson (Franklin Lakes, NJ); murine anti-human β1 integrin Ab (clone P5D2) and murine anti-human P-selectin Ab (clone P8G6) were obtained from Chemicon (Temecula, CA); murine anti-human E-selectin Ab (clone 68-5H11), murine anti-human HLA-A,B,C Ab (clone G46-2.6), and murine IgG (clone MOPC-21) were obtained from PharMingen (San Diego, CA), murine anti-human CD18 Ab was obtained from Ancell (Bayport, MN), fluorescein-conjugated goat anti-murine IgG was obtained from Organon Teknika (Durham, NC).

Blood was drawn from healthy humans by venipuncture after informed consent was obtained. Human neutrophils were isolated with Histopaque density gradients (Sigma) according to manufacturer’s protocols. The purity of isolated neutrophils was >95%.

Human pulmonary microvascular ECs were obtained from Clonetics (Walkersville, MD) and plated onto fibronectin-coated culture dishes according to manufacturer’s protocols. ECs were used between passage 6 and 10. All experiments were performed using cells 3–6 days after they reach confluence. These cells constitutively express ICAM-1, and they can be induced to express E-selectin and up-regulate ICAM-1 expression on TNF-α stimulation (13).

ECs treated with buffer or TNF-α for 24 h were studied. Cells were cultured in 96-well plates that contained ∼40,000 cells/well when the cells reached confluence. For each of the experiments, an average response from all the cells in a cell well was recorded, and the n values in this study refer to the number of individual experiments. After the baseline biomechanical properties or dichlorofluorescein (DCF) fluorescence of ECs were measured, neutrophils (neutrophil:EC, 1:1) or buffer were added to ECs, and these parameters as well as neutrophil shape were measured after 2, 6, 10, and 15 min of neutrophil adhesion. In many studies, neutrophils or ECs were pretreated with agents before the adhesion of neutrophils as described in Results to examine the mechanisms important in the observations.

The biomechanical properties of ECs were measured by magnetic twisting cytometry. This technique measures the angular rotation of ferromagnetic beads bound to cells through specific ligands on application of a magnetic torque (stress). The degree of angular rotation is inversely proportional to the stiffness of the cells to which the ferromagnetic beads are bound. Ferromagnetic beads coated with goat anti-mouse IgG (Fc) were obtained from Spherotech (Libertyville, IL). These beads were incubated with a murine Ab against human β1 integrin at a concentration of 1 μg/106 beads for 30 min at 4°C, followed by three washes in PBS. ECs treated with 20 ng/ml TNF-α or buffer for 24 h at 37°C were washed twice with DMEM containing 5% FBS and incubated with anti-β1 integrin Ab-coated beads at 37°C for 30 min. The unbound beads were gently washed off, and the well was placed in the magnetic twisting cytometer. As previously described (11, 12), the bound beads were exposed to a brief (10-μs) but strong (1000-gauss) magnetic field, which magnetizes the beads in the horizontal direction. After 20 s, the beads were twisted by a much weaker (30-gauss) but continuous (1-min) vertical magnetic field. This twisting field was not strong enough to remagnetize the beads, but it caused the beads to rotate. The magnitude of magnetic vector in the horizontal direction (remnant magnetic field) was measured by an in-line magnetometer. From this value, the average bead rotation (angular strain) was calculated (11, 12). The rotational stress was calculated by rotating the beads in a viscous standard. For these beads, a twisting field of 10 gauss corresponded to an applied torque at the start of the twist (initial stress) of 7 dynes/cm2. The specific torque (stress) on the beads at the end of the 1-min twist (stress1 min) was calculated with the use of the initial stress times the ratio of remnant field at the end of the 1-min twist and the remnant field at time 0. The apparent stiffness was measured at 1 min of twist and was defined as the ratio of stress1 min to the angular strain at this time point.

The expression of EC adhesion molecules was quantified by ELISA as previously described (26). Briefly, ECs treated with buffer or 20 ng/ml TNF-α were fixed with 0.1% paraformaldehyde for 20 min at room temperature and washed. The cells were incubated with the blocking solution containing 1% BSA and 1% goat serum for 30 min, after which the cells were incubated with 10 μg/ml primary Ab against ICAM-1, P-selectin, E-selectin, or isotype control Ab diluted in blocking solution for 1 h. The cells were washed and incubated in 10 μg/ml fluorescein-conjugated secondary Ab for 1 h. After three washes, the fluorescence (excitation wavelength, 490 nm; emission wavelength, 530 nm) was quantified using a fluorescent plate reader.

ICAM-1 was cross-linked as previously described (14). Briefly, 24-h TNF-α- or buffer-treated ECs were washed once with DMEM containing 5% FBS and incubated with 15 μg/ml murine anti-human ICAM-1 Ab (clone 6.5B5), murine anti-human HLA-A,B,C Ab (clone G46-2.6), or murine IgG for 30 min. The cells were washed twice, and rabbit anti-murine IgG Ab was added at 1:100 dilution from the manufacturer’s stock. The apparent stiffness of ECs was measured 2–15 min later.

Isolated neutrophils were labeled with sodium [51Cr]chromate as previously described (15). Confluent ECs plated onto 96-well plates were treated with 20 ng/ml TNF-α for 24 h. After two washes, ECs were incubated with 50 μg/ml murine anti-human ICAM-1 Ab (clone RR1/1) or murine IgG for 30 min and washed twice with HBSS containing 1.2 mM Ca2+, 0.4 mM Mg2+, and 5.5 mM glucose. To examine the role of CD18 in mediating neutrophil adhesion, neutrophils were incubated with 10 μg/ml anti-CD18 Ab (clone IB4) for 20 min before being added to ECs. 51Cr-Labeled neutrophils (neutrophil:EC, 1:1) were then added to the EC wells or to plastic wells containing 100 μl HBSS along with 1.2 mM Ca2+, 0.4 mM Mg2+, and 5.5 mM glucose and allowed to adhere for 15 min. Nonadherent neutrophils were then washed off, and the fraction of neutrophils that remained adherent was calculated.

ECs were grown to confluence on glass coverslips and treated with 20 ng/ml TNF-α or buffer for 24 h. Neutrophils were added to ECs and incubated for 6, 15, and 30 min. The cells were fixed and examined under a microscope with a drawing tube. To examine the projected area and shape of neutrophils, the neutrophils were outlined on a digitizing pad interfaced with a computer equipped with SigmaScan software. The projected area, as well as the length of the major axis and the axis perpendicular to the major axis at its midpoint (the minor axis), was measured for each cell. The ratio of the major to minor axis was calculated to evaluate the changes in neutrophil shape that occurred over time.

To evaluate ROS production in neutrophils, isolated neutrophils were incubated with 20 μM DCFDA for 30 min at room temperature and washed twice with Ca2+- and Mg2+-free HBSS. ECs treated with 20 ng/ml TNF-α or buffer for 24 h were washed twice with HBSS containing 1.2 mM Ca2+, 0.4 mM Mg2+, and 5.5 mM glucose before labeled neutrophils or buffer were added. DCF fluorescence was excited at 490 nm, and the emission was collected at 525 nm with a fluorescent plate reader. Neutrophil DCF fluorescence was measured 0–15 min after adherence to ECs. Then 1 μM fMLP or buffer was added to the well, and neutrophil DCF fluorescence was measured 2–15 min later.

To evaluate ROS production in neutrophils adherent to plastic, neutrophils were added to plastic wells containing 100 μl HBSS along with 1.2 mM Ca2+, 0.4 mM Mg2+, and 5.5 mM glucose. After the baseline DCF fluorescence was measured, 1 μM fMLP or buffer was added, and the fluorescence was measured 2–15 min later.

To evaluate oxidant production in ECs, ECs treated with 20 ng/ml TNF-α or buffer for 24 h were incubated with 20 μM DCFDA for 45 min at 37°C and washed twice with HBSS containing 1.2 mM Ca2+, 0.4 mM Mg2+, and 5.5 mM glucose. After the baseline DCF fluorescence was measured, neutrophils or buffer were added, and the DCF fluorescence was measured 2–20 min later.

The biomechanical properties of ECs were evaluated by magnetic twisting cytometry with the use of beads coated with anti-β1 integrin Ab as described previously (11, 12). β1 integrin was selected because it is a transmembrane protein that is linked to the cytoskeleton. Pulmonary microvascular ECs expressed β1 integrin as determined by ELISA, and TNF-α treatment did not alter its expression (data not shown). Preliminary studies indicated that the biomechanical properties measured with beads coated with either of two anti-β1 integrin Abs (clones JB1A and P5D2) were similar (apparent stiffness, 18.6 and 19.1 dynes/cm2, respectively). All subsequent experiments in this study were performed with clone P5D2. When beads coated with a control mouse IgG were used, the stiffness of ECs was 5.5 ± 1.3 dynes/cm2 (n = 4). These studies demonstrated that binding beads to ECs through cytoskeleton-linked β1 integrin decreased their ability to rotate compared with beads bound nonspecifically. Moreover, treatment of these ECs with 1 μg/ml cytochalasin D for 30 min reduced the apparent stiffness of ECs from 19.8 ± 0.7 dynes/cm2 to 12.0 ± 0.9 dynes/cm2 (n = 4), demonstrating that the integrity of the actin cytoskeleton contributed to the biomechanical properties of ECs measured with beads coated with anti-β1 integrin Ab.

Neutrophil adherence led to changes in the biomechanical properties of cytokine-activated ECs. Neutrophil adherence for 2 min induced an increase in the apparent stiffness of 24-h TNF-α-treated ECs when measured using ferromagnetic beads bound to β1 integrin on ECs (Fig. 1). This increase in the apparent stiffness of ECs required TNF-α treatment, because neutrophils adherent to untreated ECs did not increase EC stiffness (Fig. 1). Pretreatment of neutrophils with a blocking anti-CD18 Ab, which reduced the percentage of neutrophils adherent to 24-h TNF-α-treated ECs from 24.2 ± 2.2% to 2.0 ± 1.5%, significantly inhibited this stiffening response. Adherence of neutrophils treated with a control mouse IgG to ECs for 2, 6, and 10 min increased the EC stiffness from 22.0 ± 1.6 dynes/cm2 to 26.6 ± 1.3, 34.2 ± 3.0 and 32.3 ± 1.5 dynes/cm2, whereas on adherence of neutrophils pretreated with the anti-CD18 Ab, the EC stiffness changed from 18.9 ± 0.8 dynes/cm2 to 20.8 ± 1.4, 20.6 ± 2.5, and 22.5 ± 2.9 dynes/cm2 (n = 3).

FIGURE 1.

Changes in the apparent stiffness of ECs upon neutrophil adherence. The apparent stiffness of 24-h TNF-α-treated ECs (•) or untreated ECs (○) was measured by magnetic twisting cytometry using ferromagnetic beads bound to β1 integrin as described in Materials and Methods. After the measurement of stiffness baseline, purified neutrophils (neutrophil:EC, 1:1) were added to the well, and the EC stiffness was measured 2, 6, 10, and 15 min later. Data are expressed as means ± SEM (n = 4). ∗, p < 0.05 compared with the baseline stiffness.

FIGURE 1.

Changes in the apparent stiffness of ECs upon neutrophil adherence. The apparent stiffness of 24-h TNF-α-treated ECs (•) or untreated ECs (○) was measured by magnetic twisting cytometry using ferromagnetic beads bound to β1 integrin as described in Materials and Methods. After the measurement of stiffness baseline, purified neutrophils (neutrophil:EC, 1:1) were added to the well, and the EC stiffness was measured 2, 6, 10, and 15 min later. Data are expressed as means ± SEM (n = 4). ∗, p < 0.05 compared with the baseline stiffness.

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Treatment of pulmonary microvascular ECs with 20 ng/ml TNF-α for 8 or 24 h led to increases in ICAM-1 expression, whereas the expression of E-selectin and P-selectin was not significantly altered (Table I). The roles of ICAM-1 in both the stiffening response and the adhesion of neutrophils were thus examined with anti-ICAM-1 Abs. As shown in Fig. 2,a, 30 min pretreatment with 50 μg/ml murine anti-human ICAM-1 Ab (clone RR1/1), but not with the control murine IgG, significantly attenuated EC stiffening response induced by adherent neutrophils. Because the anti-ICAM-1 Ab had no effect on neutrophil adherence (Fig. 2 b), inhibition of the stiffening response was not due to a decrease in the numbers of neutrophils bound to the ECs. To confirm the lack of inhibition of adhesion by this anti-ICAM-1 Ab, another clone of anti-ICAM-1 Ab (LB2) was used to pretreat ECs. This Ab also did not inhibit neutrophil adherence to ECs (data not shown).

Table I.

Changes in the expression of EC adhesion molecules after TNF-α treatmenta

Duration of TNF-α Treatment (h)P-selectinE-selectinICAM-1
Negative controlb 1.00 ± 0.17   
1.40 ± 0.27 0.89 ± 0.07 1.62 ± 0.06 
0.5 1.25 ± 0.42 1.15 ± 0.14 1.21 ± 0.11 
1.30 ± 0.23 1.15 ± 0.18 1.63 ± 0.10 
1.44 ± 0.31 1.18 ± 0.02 2.64 ± 0.25 
1.81 ± 0.26 1.60 ± 0.27 6.14 ± 0.42cc 
24 2.22 ± 0.62 1.30 ± 0.16 5.90 ± 0.40cc 
Duration of TNF-α Treatment (h)P-selectinE-selectinICAM-1
Negative controlb 1.00 ± 0.17   
1.40 ± 0.27 0.89 ± 0.07 1.62 ± 0.06 
0.5 1.25 ± 0.42 1.15 ± 0.14 1.21 ± 0.11 
1.30 ± 0.23 1.15 ± 0.18 1.63 ± 0.10 
1.44 ± 0.31 1.18 ± 0.02 2.64 ± 0.25 
1.81 ± 0.26 1.60 ± 0.27 6.14 ± 0.42cc 
24 2.22 ± 0.62 1.30 ± 0.16 5.90 ± 0.40cc 
a

Treatment of pulmonary microvascular ECs with 20 ng/ml TNF-α induced a time-dependent increase in ICAM-1 expression but had no effect on the expression of P-selectin and E-selectin. The expression of these adhesion molecules was quantified by ELISA as described in Materials and Methods. Significant increase in ICAM-1 expression was observed after 8 and 24 h TNF-α treatment. The data were normalized by the values of the negative controls and were expressed as means ± SE (n ≥ 3).

b

, Negative control. The cells were stained the same as the others except that they were incubated with isotype control IgG as opposed to the indicated primary Ab.

c

c, p < 0.05 compared with the values of ECs not treated with TNF-α.

FIGURE 2.

Effect of anti-ICAM-1 Ab on the EC stiffening response induced by adherent neutrophils (a) and on neutrophil adherence to 24-h TNF-α-treated ECs (b). 24-h TNF-α-treated ECs were incubated with 50 μg/ml ICAM-1 Ab (○) or control murine IgG (•) along with anti-β1 integrin-coated beads for 30 min. The apparent stiffness before or 2, 6, 10, or 15 min after neutrophil adherence was evaluated as described before. To measure the percentage of adherent neutrophils, 24-h TNF-α-treated ECs were incubated with 50 μg/ml murine anti-human ICAM-1 Ab or control murine IgG for 30 min. The cells were then washed twice, and 51Cr-labeled neutrophils were allowed to adhere for 15 min. The percentage of adherent neutrophils was determined as described in Materials and Methods. Data are expressed as means ± SEM (n = 4). •, Mouse IgG control; ○, ICAM-1 Ab. ∗, p < 0.05 compared with the controls or with baseline stiffness.

FIGURE 2.

Effect of anti-ICAM-1 Ab on the EC stiffening response induced by adherent neutrophils (a) and on neutrophil adherence to 24-h TNF-α-treated ECs (b). 24-h TNF-α-treated ECs were incubated with 50 μg/ml ICAM-1 Ab (○) or control murine IgG (•) along with anti-β1 integrin-coated beads for 30 min. The apparent stiffness before or 2, 6, 10, or 15 min after neutrophil adherence was evaluated as described before. To measure the percentage of adherent neutrophils, 24-h TNF-α-treated ECs were incubated with 50 μg/ml murine anti-human ICAM-1 Ab or control murine IgG for 30 min. The cells were then washed twice, and 51Cr-labeled neutrophils were allowed to adhere for 15 min. The percentage of adherent neutrophils was determined as described in Materials and Methods. Data are expressed as means ± SEM (n = 4). •, Mouse IgG control; ○, ICAM-1 Ab. ∗, p < 0.05 compared with the controls or with baseline stiffness.

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To evaluate the roles of ROS in mediating the EC stiffening response induced by adherent neutrophils, various antioxidants and hydroxyl radical scavengers were used to pretreat ECs. After these agents were washed off, they were added to the cells again at the same concentrations as before the measurement of baseline stiffness. As shown in Fig. 3, a and b, treatment with 1% DMSO, a hydroxyl radical scavenger, or 0.3 mg/ml allopurinol, a xanthine oxidase inhibitor, inhibited the EC stiffening response induced by adherent neutrophils. In addition, treatment with 5 mM deferoxamine, an iron chelator, attenuated EC stiffening response (Fig. 3,c). These results suggest that production of ROS is essential for inducing EC stiffening response. On the other hand, treatment with 0.3 mM l-NMA to inhibit production of nitric oxide had no effect, suggesting that nitric oxide is not involved in this process (Fig. 3 d).

FIGURE 3.

Effect of antioxidants or oxygen-derived free radical scavengers on EC stiffening induced by neutrophil adherence. We pretreated 24-h TNF-α-treated ECs with various agents (○) or their vehicle (•) for 30 min along with the anti-β1 integrin-coated beads. The cells were then washed twice, and these agents were added to ECs again. The apparent stiffness of ECs before and 2, 6, 10, and 15 min after neutrophil adherence was evaluated as described. a, Pretreatment with 1% DMSO, a hydroxyl radical scavenger; b, Pretreatment with 0.3 mg/ml allopurinol, a xanthine oxidase inhibitor; c, Pretreatment with 5 mM deferoxamine, an iron chelator; d, Pretreatment with 0.3 mM l-NMA, a nitric oxide synthase inhibitor. Data are expressed as means ± SEM (n = 4). •, Control vehicle; ○, agent. ∗, p < 0.05 compared with the controls.

FIGURE 3.

Effect of antioxidants or oxygen-derived free radical scavengers on EC stiffening induced by neutrophil adherence. We pretreated 24-h TNF-α-treated ECs with various agents (○) or their vehicle (•) for 30 min along with the anti-β1 integrin-coated beads. The cells were then washed twice, and these agents were added to ECs again. The apparent stiffness of ECs before and 2, 6, 10, and 15 min after neutrophil adherence was evaluated as described. a, Pretreatment with 1% DMSO, a hydroxyl radical scavenger; b, Pretreatment with 0.3 mg/ml allopurinol, a xanthine oxidase inhibitor; c, Pretreatment with 5 mM deferoxamine, an iron chelator; d, Pretreatment with 0.3 mM l-NMA, a nitric oxide synthase inhibitor. Data are expressed as means ± SEM (n = 4). •, Control vehicle; ○, agent. ∗, p < 0.05 compared with the controls.

Close modal

To further evaluate the roles of ICAM-1 and oxidants in mediating the EC stiffening response upon neutrophil adherence, the effect of ICAM-1 cross-linking on the apparent stiffness of ECs was examined. As shown in Fig. 4,a, binding ICAM-1 with anti-ICAM-1 Ab (clone 6.5B5) did not alter the baseline stiffness of 24-h TNF-α-treated ECs. However, cross-linking ICAM-1 with the rabbit anti-murine IgG Ab increased EC stiffness within 2 min. This increase persisted for at least 15 min. This cross-linking-induced stiffening response required pretreatment with anti-ICAM-1 Ab, as pretreatment with either mouse IgG or murine anti-human HLA-A,B,C Ab had no effect. Cross-linking E-selectin also did not alter the apparent stiffness of TNF-α-treated ECs (data not shown), further supporting the specificity of the stiffening response upon ICAM-1 ligation. The stiffening response also required TNF-α pretreatment, as ICAM-1 cross-linking did not increase the apparent stiffness of untreated ECs (apparent stiffness of untreated ECs before or 2, 6, 10, and 15 min after ICAM-1 cross-linking: 14.9 ± 0.3, 15.1 ± 0.8, 15.8 ± 1.1, 17.5 ± 1.3, and 17.9 ± 1.0 dynes/cm2, respectively, n = 4). This increase in the EC stiffness upon ICAM-1 cross-linking was inhibited by allopurinol (Fig. 4 b), suggesting that ROS are involved in the EC stiffening response upon ICAM-1 cross-linking.

FIGURE 4.

Changes in the apparent stiffness of ECs on ICAM-1 cross-linking and the effect of allopurinol. a, 24-h TNF-α-treated ECs were incubated with 15 μg/ml ICAM-1 Ab (•), murine anti-human HLA-A,B,C Ab (⋄) or control murine IgG (○) along with anti-β1 integrin-coated beads for 30 min. The apparent stiffness before or 2–15 min after the addition of rabbit anti-murine IgG Ab was evaluated as described before. b, ECs were pretreated with 0.3 mg/ml allopurinol (○) or buffer (•) along with the anti-ICAM-1 Ab, and the apparent stiffness of ECs before or 2–15 min after the addition of the secondary Ab was measured. ∗, p < 0.05 compared with the controls.

FIGURE 4.

Changes in the apparent stiffness of ECs on ICAM-1 cross-linking and the effect of allopurinol. a, 24-h TNF-α-treated ECs were incubated with 15 μg/ml ICAM-1 Ab (•), murine anti-human HLA-A,B,C Ab (⋄) or control murine IgG (○) along with anti-β1 integrin-coated beads for 30 min. The apparent stiffness before or 2–15 min after the addition of rabbit anti-murine IgG Ab was evaluated as described before. b, ECs were pretreated with 0.3 mg/ml allopurinol (○) or buffer (•) along with the anti-ICAM-1 Ab, and the apparent stiffness of ECs before or 2–15 min after the addition of the secondary Ab was measured. ∗, p < 0.05 compared with the controls.

Close modal

The ROS required for EC stiffening may be derived from ROS originating from neutrophils and/or ECs. To determine whether neutrophils were the sources of ROS production during neutrophil-EC adhesion, neutrophils were labeled with DCFDA and the oxidation of DCF was measured. In neutrophils adherent to ECs for as long as 15 min, little change in DCF fluorescence was observed whether neutrophils were adherent to either untreated or TNF-α-treated ECs (Fig. 5,a). To determine whether neutrophils adherent to ECs are capable of producing oxidants, 1 μM fMLP or buffer was added to neutrophils that had been adherent to untreated ECs or to 24-h TNF-α-treated ECs for 15 min. Stimulation with fMLP, but not buffer, induced a significant increase in DCF fluorescence within 2 min, and this increase persisted for the 15 min duration of the study (Fig. 5 a). Together, these studies suggest that neutrophils do not produce oxidants in response to adherence to 24-hr TNF-α-treated ECs, although they can be stimulated to produce oxidants by fMLP.

FIGURE 5.

Oxidant production in neutrophils. Neutrophils were incubated with 20 μM DCFDA for 30 min at room temperature. The cells were then washed twice, and the DCF fluorescence was measured as described. a, fMLP-induced changes in DCF fluorescence in neutrophils adherent to untreated ECs or to 24-h TNF-α-activated ECs; b, fMLP-induced changes in DCF fluorescence in neutrophils adherent to uncoated polystyrene wells. Data are expressed as means ± SEM of six wells. ∗, p < 0.05 compared with the corresponding controls. ▪, □, Neutrophils adherent to 24-h TNF-α-activated ECs; ○, •, neutrophils adherent to untreated ECs.

FIGURE 5.

Oxidant production in neutrophils. Neutrophils were incubated with 20 μM DCFDA for 30 min at room temperature. The cells were then washed twice, and the DCF fluorescence was measured as described. a, fMLP-induced changes in DCF fluorescence in neutrophils adherent to untreated ECs or to 24-h TNF-α-activated ECs; b, fMLP-induced changes in DCF fluorescence in neutrophils adherent to uncoated polystyrene wells. Data are expressed as means ± SEM of six wells. ∗, p < 0.05 compared with the corresponding controls. ▪, □, Neutrophils adherent to 24-h TNF-α-activated ECs; ○, •, neutrophils adherent to untreated ECs.

Close modal

In contrast, neutrophil adherence to uncoated plastic wells induced increases in DCF fluorescence that were significantly greater than when similar numbers of neutrophils were adherent to TNF-α-pretreated ECs (3901 ± 393-unit increase during a 15-min interval vs 283 ± 15-unit increase during this period (Fig. 5,b compared with Fig. 5,a, p < 0.05). Treatment with 1 μM fMLP resulted in further increases in oxidant production (Fig. 5 b).

To determine whether the lack of oxidant production from neutrophils bound to TNF-α-treated ECs compared with plastic wells or to untreated ECs was due to decreased adhesion or adhesion-induced changes in neutrophil shape, adhesion assays, and quantification of shape changes were performed. Neutrophil adhesion assays demonstrated that the percentage of neutrophils adherent to 24-h TNF-α-treated ECs at 15 min (16.8 ± 1.0%) was higher than that of neutrophils adherent to uncoated plastic wells (7.3 ± 0.5%, p < 0.05) or to untreated ECs (6.3 ± 0.6%, p < 0.05). These data suggest that the lack of oxidant production in neutrophils adherent to 24-hr TNF-α-treated ECs was not due to less adhesion. Moreover, the lack of oxidant production was also not due to lack of neutrophil shape changes. The projected area of neutrophils adherent to 24-hr TNF-α-treated ECs (75.5 ± 7.0 μm2) was significantly larger than neutrophils bound to untreated ECs (37.4 ± 2.2 μm2, p < 0.05), and the measured increase in the ratio of major axis length to the minor axis length indicated that neutrophils adherent to 24-h TNF-α-treated ECs were more elongated than neutrophils bound to untreated ECs (1.65 ± 0.08 vs 1.22 ± 0.02, p < 0.05). Together, these data suggest that neutrophils adherent to 24-h TNF-α-treated ECs were well adhered, spread, and elongated but did not make ROS until stimulated by fMLP.

To determine whether ECs are the sources of ROS production, the oxidant production in ECs was measured by labeling ECs with DCFDA. In untreated ECs, the addition of neutrophils or buffer resulted in very similar increases in the DCF fluorescence over 20 min (Fig. 6,a). In contrast, in 24-h TNF-α-treated ECs, addition of neutrophils induced a greater increase in DCF fluorescence than addition of buffer (Fig. 6,b). This effect was apparent by 2 min and persisted for at least 20 min. These data suggest that adherent neutrophils induced an increase in oxidant production in 24-h TNF-α-treated, but not in untreated, ECs. This increase in oxidant production was significantly attenuated by treating ECs with 0.3 mg/ml allopurinol (Table II) but not by treating ECs with 0.3 mM l-NMA (Table III), suggesting that activation of xanthine oxidase was partially responsible for the oxidant production in 24-h TNF-α-treated ECs upon neutrophil adherence.

FIGURE 6.

Oxidant production in ECs on neutrophil adherence. Untreated ECs or TNF-α-activated ECs were loaded with DCFDA and washed. After the baseline DCF fluorescence was measured, neutrophils or buffer were added, and the DCF fluorescence was measured 2–20 min later. Data are presented as percentage changes in DCF fluorescence over the baseline DCF fluorescence before the addition of neutrophils or buffer. a, Percentage changes in DCF fluorescence in untreated ECs; b, percentage changes in DCF fluorescence in 24-h TNF-α-treated ECs. Data are expressed as means ± SEM of six wells. ∗, p < 0.05 compared with the corresponded controls. ○, Addition of buffer; •, addition of neutrophils.

FIGURE 6.

Oxidant production in ECs on neutrophil adherence. Untreated ECs or TNF-α-activated ECs were loaded with DCFDA and washed. After the baseline DCF fluorescence was measured, neutrophils or buffer were added, and the DCF fluorescence was measured 2–20 min later. Data are presented as percentage changes in DCF fluorescence over the baseline DCF fluorescence before the addition of neutrophils or buffer. a, Percentage changes in DCF fluorescence in untreated ECs; b, percentage changes in DCF fluorescence in 24-h TNF-α-treated ECs. Data are expressed as means ± SEM of six wells. ∗, p < 0.05 compared with the corresponded controls. ○, Addition of buffer; •, addition of neutrophils.

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Table II.

Effect of allopurinol on oxidant production in ECs induced by neutrophil adherencea

Time (min)Vehicle for AllopurinolAllopurinol
BufferPMNBufferPMN
52 ± 2 62 ± 3b 44 ± 2 54 ± 7 
78 ± 3 104 ± 4b ,c 66 ± 3 83 ± 6 
10 109 ± 4 150 ± 5b ,c 92 ± 3 116 ± 7b 
15 145 ± 5 203 ± 7b ,c 122 ± 4 154 ± 8b 
20 187 ± 7 257 ± 8b ,c 155 ± 5 190 ± 12b 
Time (min)Vehicle for AllopurinolAllopurinol
BufferPMNBufferPMN
52 ± 2 62 ± 3b 44 ± 2 54 ± 7 
78 ± 3 104 ± 4b ,c 66 ± 3 83 ± 6 
10 109 ± 4 150 ± 5b ,c 92 ± 3 116 ± 7b 
15 145 ± 5 203 ± 7b ,c 122 ± 4 154 ± 8b 
20 187 ± 7 257 ± 8b ,c 155 ± 5 190 ± 12b 
a

Pretreatment of ECs with allopurinol attenuated oxidant production in 24-h TNF-α-treated ECs after neutrophil adherence. TNF-α-activated ECs were incubated with DCFDA along with 0.3 mg/ml allopurinol or its corresponding vehicle for 30 min and washed. After the baseline DCF fluorescence was measured, neutrophils or buffer were added, and the DCF fluorescence was measured 2–20 min later. Data are presented as percentage changes in DCF fluorescence over the baseline DCF fluorescence before the addition of neutrophils or buffer. Data are expressed as means ± SEM of four to six wells.

b

, Significantly higher when compared with the corresponding controls in which buffer instead of neutrophils were added (p < 0.05).

c

, Significantly higher when compared with neutrophil-induced changes in DCF fluorescence in ECs pretreated with allopurinol (p < 0.05).

Table III.

Effect of l-NMA on oxidant production in ECs induced by neutrophil adherencea

Time (min)Vehicle for l-NMAl-NMA
BufferPMNBufferPMN
7 ± 1 20 ± 2b 5 ± 1 15 ± 2b 
26 ± 3 54 ± 5b 22 ± 1 48 ± 2b 
10 42 ± 4 84 ± 7b 39 ± 2 73 ± 4b 
15 58 ± 4 117 ± 11b 55 ± 3 100 ± 7b 
20 82 ± 7 162 ± 17b 82 ± 4 141 ± 9b 
Time (min)Vehicle for l-NMAl-NMA
BufferPMNBufferPMN
7 ± 1 20 ± 2b 5 ± 1 15 ± 2b 
26 ± 3 54 ± 5b 22 ± 1 48 ± 2b 
10 42 ± 4 84 ± 7b 39 ± 2 73 ± 4b 
15 58 ± 4 117 ± 11b 55 ± 3 100 ± 7b 
20 82 ± 7 162 ± 17b 82 ± 4 141 ± 9b 
a

Pretreatment of ECs with l-NMA had no effect on oxidant production in 24-h TNF-α-treated ECs after neutrophil adherence. TNF-α-activated ECs were incubated with DCFDA along with 0.3 mM l-NMA or its corresponding vehicle for 30 min and washed. After the baseline DCF fluorescence was measured, neutrophils or buffer were added, and the DCF fluorescence was measured 2–20 min later. Data are presented as percentage changes in DCF fluorescence over the baseline DCF fluorescence before the addition of neutrophils or buffer. Data are expressed as means ± SEM of four to six wells.

b

, Significantly higher when compared with the corresponding controls in which buffer instead of neutrophils were added (p < 0.05).

To examine the roles of ICAM-1 in oxidant production in ECs on neutrophil adherence, 24-h TNF-α-treated ECs were treated with 50 μg/ml murine anti-human ICAM-1 Ab (clone RR1/1) for 30 min. Treatment with this Ab, but not mouse IgG, inhibited the increase in EC ROS production on neutrophil adherence (Table IV). These data suggest that ICAM-1-mediated signaling events were involved in the oxidant production in 24-h TNF-α-treated ECs on neutrophil adherence.

Table IV.

Effect of anti-ICAM-1 Ab on oxidant production in ECs induced by neutrophil adherencea

Time (min)Mouse IgGAnti-ICAM-1 Ab
BufferPMNBufferPMN
10 ± 3 14 ± 2 10 ± 1 9 ± 1 
12 ± 2 22 ± 1b 15 ± 2 18 ± 2 
10 22 ± 2 35 ± 3b 24 ± 2 26 ± 3 
15 31 ± 1 47 ± 3b 33 ± 1 40 ± 3 
20 42 ± 1 58 ± 6b 47 ± 5 52 ± 7 
Time (min)Mouse IgGAnti-ICAM-1 Ab
BufferPMNBufferPMN
10 ± 3 14 ± 2 10 ± 1 9 ± 1 
12 ± 2 22 ± 1b 15 ± 2 18 ± 2 
10 22 ± 2 35 ± 3b 24 ± 2 26 ± 3 
15 31 ± 1 47 ± 3b 33 ± 1 40 ± 3 
20 42 ± 1 58 ± 6b 47 ± 5 52 ± 7 
a

Pretreatment of ECs with anti-ICAM-1 Ab attenuated oxidant production in 24-h TNF-α-treated ECs after neutrophil adherence. TNF-α-activated ECs were incubated with DCFDA along with 50 μg/ml anti-ICAM-1 Ab or control mouse IgG for 30 min and washed. After the baseline DCF fluorescence was measured, neutrophils or buffer were added, and the DCF fluorescence was measured 2–20 min later. Data are presented as percentage changes in DCF fluorescence over the baseline DCF fluorescence before the addition of neutrophils or buffer. Data are expressed as means ± SEM of four to six wells.

b

, Significantly higher when compared with the corresponding controls in which buffer instead of neutrophils were added (p < 0.05).

This study demonstrates that neutrophil adherence to 24-h TNF-α-activated human pulmonary microvascular ECs, but not to untreated ECs, induced an increase in the apparent stiffness of ECs within 2 min of adhesion. This stiffening response induced by neutrophils was inhibited by an anti-ICAM-1 Ab and was mimicked by cross-linking ICAM-1, suggesting that ICAM-1-mediated signaling events may be involved. The neutrophil-induced EC stiffening response was inhibited by DMSO, a hydroxyl radical scavenger, allopurinol, a xanthine oxidase inhibitor, or deferoxamine, an iron chelator, but not with l-NMA, a nitric oxide synthase inhibitor. These data suggest that hydrogen peroxide and iron-dependent hydroxyl radicals derived from xanthine oxidase-generated superoxide are involved in mediating the EC stiffening response. Neutrophils adherent to 24-h TNF-α-activated ECs produced almost nondetectable amounts of ROS, although they responded to fMLP by generating ROS. However, neutrophil adherence induced ROS production in 24-h TNF-α-activated ECs within 2 min, similar to the time required for the stiffening response. The production of EC ROS was largely derived from a xanthine oxidase-catalyzed reaction in ECs. The ICAM-1 Ab prevented the ROS production in ECs. Taken together, on adherence of neutrophils, ICAM-1 may signal into ECs to produce ROS and initiate biomechanical changes in ECs.

In this study, we used anti-β1 integrin Ab-coated beads to probe changes in the biomechanical properties of ECs on neutrophil adherence. Previous studies have used beads coated with synthetic RGD peptide or anti-β1 integrin Ab to evaluate the biomechanical properties of cultured ECs (18). The results show similar apparent stiffness measurements that are cytochalasin d-sensitive when either type of beads was used, suggesting that β1 integrin is an effective molecule for coupling the mechanical stress to the cytoskeleton (18). In addition, the expression of β1 integrin on the EC surface was unaltered by TNF-α treatment, which allowed us to compare the apparent stiffness measurement of untreated ECs and TNF-α-treated ECs without the confounding effects of increased β1 integrin density on bead binding.

Neutrophil adherence induced changes in the biomechanical properties of 24-h TNF-α-treated pulmonary microvascular ECs that were ICAM-1 dependent. The ability of ICAM-1 to function as a signaling molecule has been demonstrated in previous studies (14, 16, 17, 18, 19, 20). In cultured ECs, ICAM-1 cross-linking induces increases in intracellular Ca2+, activation of ErK-1 and AP-1 transcription factor, as well as up-regulation of ICAM-1 and VCAM-1 mRNA (14, 17). Interestingly, these responses induced by ICAM-1 cross-linking mimic the responses induced by leukocyte adhesion (17). Moreover, ICAM-1 activation by a mAb induces activation of Rho, tyrosine kinase p60src, and tyrosine phosphorylation of an actin cross-linking protein, cortactin, in rat microvascular ECs (18, 19, 20). The interaction of cortactin with F-actin is regulated by Src-mediated tyrosine phosphorylation (21), and cortactin has been implicated in the regulation of EC locomotion (22). Thus, neutrophil adherence through ICAM-1 ligation induces signaling events in ECs, which may ultimately modulate cytoskeleton organization.

Despite the inhibitory effects of the ICAM-1 Ab (clone RR1/1) on EC stiffening response induced by neutrophils, this Ab had no effect on neutrophil adherence to 24-h TNF-α-treated ECs. This is in contrast to a previous report (26) in which the same anti-ICAM-1 Ab partially inhibited neutrophil adherence to human umbilical vein ECs stimulated with IL-1 for 4 h. Similar results were obtained with another anti-ICAM-1 Ab (clone LB2). The basis for this discrepancy remains to be determined. Neutrophil adherence to 24-h TNF-α-treated ECs required CD18, because an anti-CD18 Ab completely inhibited neutrophil adhesion. Thus, neutrophil adhesion to these cultured pulmonary microvascular ECs appears to be mediated through CD11/CD18 binding to ligands other than ICAM-1. These possible ligands include fibronectin, fibrinogen, and ICAM-2.

Our studies suggest that ICAM-1 signaling involves activation of xanthine oxidase and production of ROS. An anti-ICAM-1 Ab inhibited oxidant production in ECs as well as the EC stiffening response upon neutrophil adherence. In addition, the EC stiffening response induced by ICAM-1 cross-linking was inhibited by allopurinol. Activation of EC xanthine oxidase by neutrophils has been reported by several investigators. For instance, adherence of PMA-activated neutrophils to untreated ECs induces activation of xanthine oxidase in ECs, and neutrophil-derived hydrogen peroxide is not sufficient to induce this effect (23). In addition, leukotriene B4, thrombin, or PMA-induced neutrophil adherence to untreated ECs induces an increase in EC xanthine oxidase activity which is not inhibited by protease inhibitors and is not mimicked by exogenous hydrogen peroxide or neutrophil elastase (24). The importance of ICAM-1-induced signaling pathways in the activation of xanthine oxidase is supported by studies demonstrating that anti-ICAM-1 Ab completely blocks conversion of xanthine oxidase from the inactive xanthine dehydrogenase in ECs induced by neutrophil adherence in response to PMA (24, 25). These observations together with the studies presented in this paper suggest that activation of EC xanthine oxidase by adherent neutrophils may occur as the result of the signaling events induced by engagement of ICAM-1.

The inhibitory effect of allopurinol on the oxidant production was only partial, suggesting that other sources of superoxide may also contribute. Functional NADPH oxidase is expressed in cultured microvascular ECs (27), and membrane-associated NADPH oxidase activation in pulmonary endothelium is an important source of oxidants in lungs during ischemia injury (28). The roles of NADPH oxidase in mediating oxidant production in ECs after neutrophil adherence remain to be determined. Superoxide production by mitochondrial enzymes may also contribute. Our studies do exclude a role for nitric oxide in EC stiffening.

ROS generated at neutrophil adherence may modulate F-actin organization. Recent evidence indicates that ROS may activate various signal transduction pathways that are implicated in regulating F-actin organization. Our previous studies demonstrated that neutrophil adherence-induced increase in EC stiffness was completely inhibited by treating ECs with cytochalasin D, a F-actin disrupting agent, or jasplakinolide, an agent that stabilizes F-actin, suggesting that F-actin rearrangement is required for this stiffening response.5 Exogenous hydrogen peroxide activates phospholipase D (29, 30, 31), which has been shown to induce actin polymerization (32). Moreover, hydrogen peroxide induces phosphatidylinositol 4,5-biphosphate formation in ECs (31), and phosphatidylinositol 4,5-biphosphate regulates actin remodeling by acting on several actin-binding proteins including gelsolin, vinculin, profilin, and α-actinin (33, 34, 35, 36). ROS also act as intracellular second messengers and have been implicated in the signaling cascades in response to growth factors and cytokines (37, 38, 39). Thus, intracellular ROS, as second messengers, may induce downstream signaling pathways that modulate the actin cytoskeleton. How these signaling events are transduced in ECs on neutrophil adherence is unclear. Changes in DCF fluorescence allow us to assess only the gross changes in oxidant production in the cells. Other investigators have postulated that the generation of oxidants inside the cells may be highly localized, and the site of production may be critical for the effect of oxidants on signaling pathways (37, 38, 39).

Although the ROS mediating EC stiffening response could be formed from neutrophil-derived ROS and/or from EC-derived ROS, this study clearly shows that ROS generated during neutrophil adhesion to TNF-α-activated ECs were not derived from neutrophils. In contrast, neutrophils adherent to untreated ECs or uncoated plastic wells did produce DCF-oxidizing ROS in response to fMLP stimulation (Fig. 5,a compared with Fig. 5 b). This lack of ROS production was not due to less adherence, because the percentage and the number of neutrophils adherent to 24-h TNF-α-activated ECs were higher than those adherent to the uncoated plastic wells. In addition, this difference was not due to global inhibition of neutrophil activation by TNF-α-activated ECs, because fMLP induced radical production in both EC- and plastic-adherent neutrophils. Finally, the lack of ROS was not due to lack of neutrophil shape changes or cytoskeletal rearrangements, because neutrophils became spread and elongated when adherent to TNF-α-activated ECs but not untreated ECs. Thus, neutrophil spreading and F-actin reorganization upon adherence to 24-h TNF-α-activated ECs are not sufficient to generate ROS in neutrophils.

This study clearly demonstrates that neutrophil adhesion to ECs induces signaling pathways in ECs through ligation of ICAM-1 that result in stiffening of these cells. The function of this stiffening, as well as its molecular mechanisms, is not yet clear. One possibility is that it represents the first phase of neutrophil-induced EC injury and death. However, a more likely possibility is that changes in the actin cytoskeleton modulate EC shape and surface characteristics that facilitate neutrophil migration toward the EC junctions. Cytoskeletal changes may also alter the functions of paracellular junctions. Recent studies from our laboratory have demonstrated that inhibiting this cytoskeletal stiffening does in fact prevent neutrophil migration to the borders of ECs. Finally, cytoskeletal reorganization may regulate signaling pathways by modulating their cytoplasmic compartmentalization. These changes in ECs induced by adherent neutrophils may contribute to EC permeability changes and modulate neutrophil emigration through the EC junctions in inflammation.

We thank Jeffrey Fredberg for his expertise in magnetic twisting cytometry and Amy Imrich and Lester Kobzik for their help in the measurement of ROS.

1

This work was supported by National Institutes of Health Grants HL 48160 and HL 33009 and a Clinical Scientist Award in Transitional Research from the Burroughs Wellcome Fund (to C.M.D).

4

Abbreviations used in this paper: ECs, endothelial cells; l-NMA, N-methyl-l-arginine; ROS, reactive oxygen species; DCF, dichlorofluorescein; DCFDA, 2′,7′-dichlorofluorescein diacetate.

5

Q. Wang, E. T. Chiang, M. Lim, J. Lai, R. Rogers, P. A. Janmey, D. Shepro, and C. M. Doerschuk. Changes in the biomechanical properties of neutrophils and endothelial cells during adhesion. Submitted for publication.

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