Activation of vascular endothelium plays an essential role in vasoocclusion in sickle cell disease. The anti-inflammatory agents dexamethasone and adhesion molecule-blocking antibodies were used to inhibit endothelial cell activation and hypoxia-induced vasoocclusion. Transgenic sickle mice, expressing human α-, βS-, and βS-Antilles-globins, had an activated vascular endothelium in their liver, lungs, and skin, as exhibited by increased activation of NF-κB compared with normal mice. NF-κB activation increased further in the liver and skin after sickle mice were exposed to hypoxia. Sickle mice had decreases in red blood cell (RBC) velocities and developed vasoocclusions in subcutaneous venules in response to hypoxia. Dexamethasone pretreatment prevented decreases in RBC velocities and inhibited vasoocclusions and leukocyte-endothelium interactions in venules after hypoxia. Dexamethasone treatment inhibited NF-κB, VCAM-1, and ICAM-1 expression in the liver, lungs, and skin of sickle mice after hypoxia-reoxygenation. VCAM-1 or ICAM-1 blockade with monoclonal antibodies mimicked dexamethasone by inhibiting vasoocclusion and leukocyte adhesion in sickle mice, demonstrating that endothelial cell activation and VCAM-1 and ICAM-1 expression are necessary for hypoxia-induced vasoocclusion in sickle mice. VCAM-1, ICAM-1, and vasoocclusion increased significantly 3 days after dexamethasone discontinuation, possibly explaining rebounds in vasoocclusive crises observed after withdrawal of glucocorticosteroids in sickle patients. We conclude that anti-inflammatory treatments that inhibit endothelial cell activation and adhesion molecule expression can inhibit vasoocclusion in sickle cell disease. Rebounds in vasoocclusive crises after dexamethasone withdrawal are caused by rebounds in endothelial cell activation.
- vascular cell adhesion molecule
- intercellular adhesion molecule
- nuclear factor-κB
- sickle cell anemia
in sickle cell disease, a single point mutation in the β-chain of hemoglobin causes hemoglobin polymerization and red blood cell (RBC) shape changes during hemoglobin deoxygenation (11, 40). This mutation leads to impaired microvascular blood flow (22), episodic vasoocclusion (18), ischemia-reperfusion injury pathophysiology (38), and endothelial cell activation (6, 45).
Endothelial cells can be activated by a diverse number of inflammatory stimuli such as oxidative stress (including hypoxia), proinflammatory cytokines, sickle RBC, and infections (10, 44, 47, 49). Glucocorticosteroids are potent anti-inflammatory drugs that inhibit endothelial cell activation (16). Studies demonstrating a beneficial effect of glucocorticosteroid therapy for pain episodes and acute chest syndrome in sickle cell disease provide indirect evidence for the role of inflammation and endothelial cell activation in vasoocclusion (8, 24). High-dose glucocorticosteroid therapy reduced the duration of hospitalization in children with vasoocclusive crisis as well as in children with mild to moderately severe acute chest syndrome (8, 24). However, these children tended to have rebounds in vasoocclusive pain crises after withdrawal of glucocorticosteroid therapy.
Glucocorticosteroids inhibit inflammation and thus endothelial cell activation through an interaction of glucocorticoid receptors with the p65 subunit of NF-κB (16, 36). Glucocorticosteroids also induce expression of IκBα, which maintains NF-κB in an inactive cytoplasmic complex (16). Similar to NF-κB activation, activator protein-1 (AP-1)-mediated activation of inflammatory genes is repressed by glucocorticosteroids through mutually negative interactions between AP-1 and glucocorticoid receptors (16, 30). Because of these anti-inflammatory properties, glucocorticosteroids inhibit the expression of P-selectin, VCAM-1, and ICAM-1 on endothelium (4, 28, 37).
Tissue hypoxia is common in patients with sickle cell disease (22, 53), especially in venular beds, where flow is sluggish and deoxygenation of hemoglobin S is favored. Systemic hypoxia in rodents induces a rapid microvascular inflammatory response characterized by the production of reactive oxygen species (ROS), activation of vascular endothelium, increased leukocyte-endothelium rolling, adhesion and emigration, and enhanced vascular permeability (12, 31, 57). P-selectin plays a prominent role in the endothelium’s early response to hypoxia, as hypoxia stimulates ROS production, which triggers cell surface expression of preformed P-selectin found inside endothelial cell Weibel-Palade bodies (41, 48). P-selectin expression on the endothelial cell surface induces leukocyte rolling, leading to firm adhesion by VCAM-1 and ICAM-1 (46). P-selectin blockade or P-selectin knockout in transgenic sickle mice inhibits hypoxia- or TNF-α-induced interactions of sickle leukocytes with the endothelium and normalizes venular blood flow (31, 51). The inflammatory response to hypoxia is accentuated in transgenic sickle mice compared with normal mice and leads to reduced blood flow and transient stasis in venules of sickle mice but not normal mice (29, 31).
In this study, systemic hypoxia was used to induce sickling, an inflammatory response, and vasoocclusion in transgenic sickle mice. The anti-inflammatory agents dexamethasone and adhesion molecule-blocking antibodies were used to examine the relationship among endothelial cell activation, adhesion molecule expression, and hypoxia-induced impairment of venular blood flow. We examined whether inhibition of inflammation with either dexamethasone or VCAM-1 or ICAM-1 blockade could inhibit hypoxia-induced vasoocclusion in transgenic sickle mice and whether the clinically recognized rebound phenomenon seen after glucocorticosteroid withdrawal was associated with an increase in vasoocclusion and in VCAM-1 and ICAM-1 expression.
All animal experiments were approved by the University of Minnesota’s Institutional Animal Care and Use Committee. We used male and female S+S-Antilles transgenic sickle mice as our mouse model for sickle cell disease (20). The S+S-Antilles mice are homozygous for deletion of the mouse β major globin locus and express human α-, βS-, and βS-Antilles-globin transgenes. βS-Antilles-globin contains, in addition to the βS mutation at β6, a second mutation at β23 (Val→Ile). βS-Antilles has low oxygen affinity and decreased solubility under deoxygenated conditions, resulting in a more severe form of sickle cell disease. In the S+S Antilles mice, ∼42% of the β-globins expressed are βS and 36% are βS-Antilles. These transgenic mice are on the C57BL/6 genetic background.
Normal male and female mice (C57BL/6) obtained from Jackson Laboratory (Bar Harbor, ME) were used as controls for the sickle mice. The mice used in these studies were 8–12 wk of age. The mice weighed 20–30 g and were housed in specific pathogen-free housing to prevent common murine infections that could cause an inflammatory response. All of the mice were maintained on a standard chow diet.
Intravital microscopy and analysis.
Dorsal skin-fold chambers (DSFC) were implanted onto mice as previously described (29). All measurements of blood flow parameters in the DSFC were made 4–7 days after DSFC implantation. To visualize blood flow, we placed anesthetized sickle mice on a specially constructed microscope stage designed to hold the mouse and align the DSFC window with the microscope optics. Intravital microscopic observations of subcutaneous venules, including mean vessel diameters, mean RBC velocities, wall shear rates, stasis/vasoocclusion, and leukocyte-endothelium interactions were carried out as previously described (29). Mean vessel diameters were measured off-line using a digital image splitting device (26). Mean RBC velocities (VRBC centerline) were measured on-line using an optical Doppler velocimeter (9) obtained from Texas A&M University Microcirculation Research Institute. Wall shear rates were calculated using the formula 8Vmean/D, where D is the vessel diameter and Vmean is the mean velocity obtained from the equation Vmean = VRBC centerline/1.6 as previously reported (15). Venules with no observable blood flow were counted as static. The percentage of static vessels was calculated by dividing the number of static venules at any given time point by the total number of venules examined.
Leukocyte-endothelium interactions were measured after leukocytes were stained in vivo with the intravital fluorescent dye rhodamine 6G as previously described (5). Rhodamine 6G was administered intravenously via tail vein injection 15 min before intravital microscopy. Leukocyte-endothelium interactions inside the venular lumen were captured on camera and recorded on videotape. Rolling leukocytes were counted off-line using the videotape and were defined as leukocytes that distinctly roll along the endothelial surface of venules as previously described (31). The rolling flux was determined as the total number of leukocytes rolling through a given section of vessel per minute. To assess leukocyte adhesion, we examined 100-μm venular segments and considered a leukocyte adherent if it remained stationary for at least 30 s (31).
At baseline, with the mice in ambient air, flowing venules were selected at random and their relative locations noted on a map of the microscopic field. Venule diameters, mean RBC velocities, stasis, leukocyte rolling, and leukocyte adhesion were measured and recorded. After baseline measurements were obtained in ambient air, the mice were transferred to a special chamber and subjected to 1 h of hypoxia (7% O2-93% N2) followed by reoxygenation in ambient air as described in Treatment with hypoxia. Measurements of blood flow parameters were repeated after 1 h of reoxygenation on the same venules that were examined at baseline.
Treatment with hypoxia.
Sickle or normal mice were placed in a special chamber and exposed to either 1 or 3 h of hypoxia (7% O2-93% N2) followed by reoxygenation in room air for the indicated times. Molecular measurements (NF-κB, VCAM-1, and ICAM-1) were made after the mice were exposed to 3 h of hypoxia and 2 h of reoxygenation, and DSFC measurements of blood flow, including vasoocclusion, were made after the mice were exposed to 1 h of hypoxia and 1 h of reoxygenation. We chose 1 h of hypoxia to allow sufficient time for all of the DSFC measurements to be made in 1 day, and 1 h of reoxygenation was chosen to permit maximal vasoocclusion in sickle mice (29). The length of hypoxia exposure was not critical to the outcome or the interpretation of the results; however, the length of the reoxygenation phase after hypoxia was important to the outcome, especially when measuring vasoocclusion, which peaked after 1 h of reoxygenation and declined thereafter.
Treatment with dexamethasone.
Sickle mice were injected intraperitoneally with 1.0 mg/kg body wt dexamethasone once per day for 3 days. This dose of dexamethasone has been previously shown to inhibit VCAM-1 and ICAM-1 expression in rats with inflamed airways (28). Bernini et al. (8) used 0.6 mg·kg−1·day−1 dexamethasone for 2 days in children with sickle cell disease. Griffin et al. (24) used 15 mg·kg−1·day−1 methylprednisolone for 2 days. Thus our dose in sickle mice was in the range used by other studies but was administered 1 day longer than has been previously published for pediatric sickle patients. Placebo sickle mice were injected intraperitoneally with sterile water. Mouse tissues were harvested, and venular blood flow parameters were measured 24 h after the third injection.
In DSFC experiments designed to measure a rebound in vasoocclusion after dexamethasone withdrawal, sickle mice were exposed to 1 h of hypoxia (7% O2-93% N2) twice: once 24 h after dexamethasone withdrawal and again 72 h after dexamethasone withdrawal. The percentage of static venules inside the DSFC was calculated 1 h after each hypoxic exposure during reoxygenation in ambient air.
VCAM-1 and ICAM-1 blockade.
Immediately after 1 h of hypoxia, at the beginning of reoxygenation, sickle mice with implanted DSFC were injected intravenously via the tail vein with 30 μg of either rat anti-mouse VCAM-1 monoclonal antibody (MAb) (clone 429), hamster anti-mouse ICAM-1 MAb (clone 3E2), or the appropriate nonspecific isotype control MAb (clones R35-95 and A19-3; BD Biosciences Pharmingen, San Diego, CA). In some control experiments, where indicated, mice were injected intravenously with 30 μg of albumin instead of nonspecific isotype control MAb. The anti-VCAM-1 and anti-ICAM-1 MAb block the binding of α4β1 and CD11/CD18 integrin counterreceptors to VCAM-1 and ICAM-1, respectively. Static venules, leukocyte rolling, and adhesion were counted by using intravital microscopy inside the DSFC (see Intravital microscopy and analysis) with and without VCAM-1 and ICAM-1 blockade.
Mouse tissue collection.
The mice were killed and tissues harvested 24 h after the last dexamethasone treatment and after exposure to 3 h of hypoxia and 2 h of reoxygenation. In the dexamethasone rebound experiments, the tissues were harvested 72 h after the last dexamethasone injection and after exposure to 3 h of hypoxia and 2 h of reoxygenation. After 2 h of reoxygenation, mice were asphyxiated in a CO2 chamber for ∼60 s. Samples of livers, lungs, and skin were taken for homogenate preparation and immunohistochemistry. Organ samples were immediately frozen in liquid nitrogen and stored at −80°C. Lung sections for immunohistochemistry were stored in tissue-freezing medium (Baxter Scientific Products, Chicago, IL).
EMSA for NF-κB activation.
Organ tissue homogenate was prepared as previously described (6, 17). Organ homogenate DNA concentrations were determined using a fluorometric DNA dye-binding assay (Bio-Rad, Hercules, CA). The bisbenzimide dye (Hoechst 33258) binds specifically to double-stranded DNA (dsDNA). RNA does not interfere significantly with the assay. Organ extracts were incubated with end-labeled 32P-dsDNA containing a consensus NF-κB DNA binding sequence (underlined bp): 5′-AGTTGAGGGGACTTTCCCAGGC-3′ (Santa Cruz Biotechnology, Santa Cruz, CA). DNA-protein binding reactions contained 100–300 ng of homogenate DNA and 70 fmol of radiolabeled NF-κB consensus dsDNA. Reactions were carried out in 20 mM HEPES (pH 7.9), 5 mM KCl, 0.5 mM EDTA, 5% glycerol, 1 mM DTT, 0.5 mM PMSF, 1 mg/ml BSA, 0.1% NP-40, and 250 ng poly(dI-dC). Binding reactions were incubated for 30 min at room temperature. Reaction mixtures were separated on a 10% nondenaturing polyacrylamide gel using 0.5× Tris-borate-EDTA running buffer. To confirm the identity of the NF-κB band, some reactions were run with an excess of unlabeled consensus or mutant dsDNA for competition experiments (Santa Cruz Biotechnology). The mutant dsDNA for NF-κB differed by 1 bp from the consensus binding sequence given above. A 10-fold excess of mutant dsDNA added to the binding reaction was unable to inhibit binding of the radiolabeled consensus dsDNA sequence to NF-κB, whereas a 10-fold excess of unlabeled consensus dsDNA completely abolished binding of the radiolabeled consensus dsDNA (data not shown). Mouse NF-κB EMSA bands contained the p50 and p65 subunits (data not shown).
Western blots of liver, lung, and skin VCAM-1 and ICAM-1.
Liver, lung, and skin tissue homogenates were prepared as described (6, 17). Tissue homogenates containing an equal amount of homogenate DNA per well were subjected to SDS-PAGE (7.5%). After SDS-PAGE, the samples were transferred electrophoretically to polyvinylidene difluoride membranes, and immunoblotting of the organ homogenates was performed with goat anti-VCAM-1 or ICAM-1 IgG directed against the COOH terminus of the target protein (Santa Cruz Biotechnology). Sites of primary antibody binding were visualized with horseradish peroxidase-conjugated donkey anti-goat IgG (Jackson ImmunoResearch Laboratories, West Grove, PA). The final detection of immunoreactive bands was performed using a chemiluminescent detection substrate (Pierce, Rockford, IL).
Quantitation of EMSA and Western blots.
Exposed films of radioactive EMSA gels and chemiluminescent Western blots were scanned on an imaging densitometer (Bio-Rad). Bands on each image were quantified with Molecular Analyst software (Bio-Rad) using local background subtraction. The intensity of VCAM-1 and ICAM-1 bands on film was linear between 25 and 1,000 ng of tissue homogenate DNA with correlation coefficients (r2) of 1.00 and 0.98, respectively. The intensity of NF-κB bands on film was linear between 12 and 500 ng of tissue homogenate DNA with an r2 of 0.98. This permitted accurate quantitation of the relative intensities of VCAM-1, ICAM-1, and NF-κB bands on film and their corresponding levels of tissue expression.
Immunohistochemistry of lung tissue.
Immunohistochemistry of lung VCAM-1 was performed as previously described (6).
All statistical analyses were performed with SigmaStat 2.0 for Windows (SPSS, Chicago, IL). Comparisons of data groups were made using a Student’s t-test or a Mann-Whitney rank sum test. Bonferroni adjustments were made to P values to adjust for multiple comparisons where appropriate. The proportions of venules exhibiting stasis in each treatment group were compared using a z-test.
Intravital microscopy and the DSFC model were used to examine the effects of dexamethasone on blood flow in the subcutaneous venules of transgenic sickle mice (Table 1). At baseline in ambient air, the mean venular diameters in the DSFC were ∼20 μm in both the placebo- and the dexamethasone-treated sickle mice, and RBC velocities and wall shear rates were similar between placebo- and dexamethasone-treated sickle mice (Table 1). After the sickle mice were exposed to 1 h of hypoxia and 1 h of reoxygenation, there were no significant changes in mean venular diameters from baseline. However, there were significant mean differences from baseline induced by hypoxia-reoxygenation in placebo-treated sickle mice in RBC velocities (P ≤ 0.001) and wall shear rates (P < 0.01; Table 1). RBC velocities and wall shear rates fell precipitously in placebo-treated sickle mice but not in dexamethasone-treated mice in response to hypoxia-reoxygenation. Thus dexamethasone treatment prevented hypoxia-reoxygenation-induced decreases in venular blood flow.
We previously reported (29) that transgenic sickle mice develop stasis and have adherent leukocytes and misshapen RBC along the endothelium of suspected static venules in the skin after hypoxia-reoxygenation. We used the DSFC and intravital microscopy to examine the effects of dexamethasone on vasoocclusion and leukocyte-endothelium interactions in subcutaneous venules of sickle mice (Table 2). At least 20 flowing subcutaneous venules were selected at random at baseline in ambient air in each placebo- and dexamethasone-treated sickle mouse. All of the flowing venules selected at baseline in ambient air were reexamined after 1 h of hypoxia and 1 h of reoxygenation for stasis (no blood flow). The percentage of static venules after hypoxia-reoxygenation was 12.0% in placebo-treated and 0.0% in dexamethasone-treated sickle mice (P < 0.001; Table 2). Approximately 50% of the venules that were static after 1 h of reoxygenation were reflowing after 4 h of reoxygenation (data not shown). Untreated normal control mice, after exposure to hypoxia, had no static vessels at any time during reoxygenation.
Leukocyte-endothelium interactions were measured after leukocytes were stained in vivo with the intravital fluorescent dye rhodamine 6G as previously described (5). The effects of dexamethasone therapy on leukocyte rolling and adhesion along the endothelium of subcutaneous venules inside the DSFC were examined in normal and sickle mice after exposure to 1 h of hypoxia and 1 h of reoxygenation. Placebo-treated sickle mice had significantly more leukocyte firm adhesion than either dexamethasone-treated sickle mice or untreated normal mice after exposure to 1 h of hypoxia and 1 h of reoxygenation (P < 0.001; Table 3). Placebo-treated sickle mice also appeared to have more leukocyte rolling than either dexamethasone-treated sickle mice or untreated normal mice, but the leukocyte rolling values did not reach statistical significance after a Bonferroni correction (0.025 < P < 0.05). Dexamethasone-treatment decreased leukocyte firm adhesion by 70% (P < 0.001) after hypoxia-reoxygenation compared with placebo-treated sickle mice (Table 3). We previously reported that there is an overabundance of firmly adherent leukocytes at sites of vascular stasis (29). Venules that become static after hypoxia-reoxygenation are filled with adherent leukocytes at bifurcations near the “head” or beginning of static venules (29). The adherent leukocytes appear to be plugging the static venules.
Because leukocyte adhesion to endothelium appeared to be associated with hypoxia-induced vasoocclusion in transgenic sickle mice, we examined the effects of leukocyte adhesion molecule blockade on vasoocclusion. We measured hypoxia-induced stasis in sickle mice with DSFCs after VCAM-1 or ICAM-1 blockade with MAbs or their isotype controls. Remarkably, after 1 h of hypoxia and 1 h of reoxygenation, stasis in sickle mice was completely prevented by either anti-VCAM-1 (P < 0.001) or anti-ICAM-1 (P < 0.01) MAb (Table 4). Isotype control antibodies were unable to prevent stasis and, in the case of the VCAM-1 isotype control, which was a rat MAb, may have worsened vasoocclusion.
Because adhesion molecule blockade inhibits hypoxia-reoxygenation-induced vasoocclusion and clumps of adherent leukocytes have been previously seen in static venules after hypoxia-reoxygenation (29), we examined the effect of adhesion molecule blockade on hypoxia-reoxygenation-induced leukocyte adhesion in the venules of sickle mice with DSFCs. Intravenous anti-VCAM-1 or anti-ICAM-1 MAbs, but not intravenous albumin, completely inhibited hypoxia-reoxygenation-induced increases in the number of adherent leukocytes in the venules of sickle mice with DSFCs (P < 0.05; Fig. 1). As expected, intravenous treatment with anti-VCAM-1, anti-ICAM-1, or albumin did not inhibit hypoxia-reoxygenation-induced leukocyte rolling in the same venules (data not shown).
Because endothelial cell adhesion molecules appeared to play an important role in hypoxia-reoxygenation-induced vasoocclusion in transgenic sickle mice, we examined the activation state of NF-κB in organs of normal and sickle mice before and after exposure to hypoxia-reoxygenation (Fig. 2). NF-κB activation was significantly increased in the liver (582%, P ≤ 0.001), lungs (322%, P < 0.01), and skin (201%, P < 0.05) of sickle mice compared with normal control mice in ambient air. After hypoxia-reoxygenation, NF-κB activation in sickle mice was further increased by 50% in the both the liver (150% of sickle mouse in ambient air, P < 0.05) and the skin (154% of sickle mouse in ambient air, P < 0.05) but not in the lungs (93% of sickle mouse in ambient air). Exposure to hypoxia-reoxygenation had no significant effects on NF-κB activation in any organs of normal control mice.
Because dexamethasone pretreatment inhibited hypoxia-induced decreases in venular blood flow, increases in leukocyte adhesion, and vasoocclusion in sickle mice (Tables 1–3), and adhesion molecule blockade also inhibited vasoocclusion and leukocyte adhesion in sickle cell mice (Table 4 and Fig. 1), we hypothesized that dexamethasone was preventing vasoocclusion in sickle mice by inhibiting NF-κB-mediated adhesion molecule expression. The effects of dexamethasone on markers of endothelial cell activation were examined in the liver, lungs, and skin of sickle mice after exposure to hypoxia-reoxygenation. Dexamethasone treatment of sickle mice, compared with placebo, reduced NF-κB activation (Fig. 3) in the liver by 51% (P < 0.01), in the lungs by 60% (P < 0.001), and in the skin by 47% (P < 0.01).
Because NF-κB activation in the liver, lungs, and skin of sickle mice was inhibited by dexamethasone treatment, we examined the effects of dexamethasone on adhesion molecule expression in the same organs. Dexamethasone treatment of sickle mice reduced mean VCAM-1 expression (Fig. 4) in the liver by 61% (P < 0.05), in the lungs by 46% (P < 0.01), and in the skin by 66% (P ≤ 0.001) compared with placebo treatment of sickle mice after hypoxia-reoxygenation.
To confirm these findings, we examined VCAM-1 expression in the lungs by using immunohistochemistry after hypoxia-reoxygenation in placebo- and dexamethasone-treated sickle mice (Fig. 5). Compared with placebo (Fig. 5, left), VCAM-1 staining is decreased in the pulmonary veins of dexamethasone-treated sickle mice (Fig. 5, right). We previously reported the colocalization of virtually all pulmonary vein VCAM-1 with von Willebrand factor in sickle mice (6), verifying the endothelial location of VCAM-1.
Similar to NF-κB and VCAM-1 expression, mean ICAM-1 expression after dexamethasone treatment of sickle mice (Fig. 6) was reduced in the lungs by 45% (P < 0.01) and in the skin by 37% (P < 0.05) compared with placebo treatment of mice after hypoxia-reoxygenation. In contrast, dexamethasone treatment had no effect on mean ICAM-1 expression in the liver after hypoxia-reoxygenation.
Because adhesion molecules play a key role during hypoxia-induced vasoocclusion in transgenic sickle mice, and because sickle cell patients often experience a rebound in clinical crises after glucocorticosteroid therapy is discontinued, we wondered whether dexamethasone withdrawal would cause a rebound in vasoocclusion and adhesion molecule expression in sickle mice. Sickle mice with implanted DSFCs were treated with dexamethasone (1 mg/kg for 3 days) or placebo. Hypoxia-reoxygenation-induced vasoocclusion in DSFC venules was measured 24 h and 72 h after the last dexamethasone or placebo injection. On both days, stasis was measured in the same venules after 1 h of hypoxia and 1 h of reoxygenation (Fig. 7). Placebo-treated sickle mice had similar percentages of stasis 24 h (19%) and 72 h (12%) after the last placebo injection (P < 0.12, stasis at 24 vs. 72 h in placebo-treated sickle mice). In contrast, dexamethasone-treated sickle mice had a dramatic increase in the percentage of hypoxia-reoxygenation-induced stasis from 24 h (0%) to 72 h (60%) after dexamethasone withdrawal (P < 0.001, stasis at 24 vs. 72 h in dexamethasone-treated sickle mice). After hypoxia-reoxygenation at 24 h, none of the venules became static in the dexamethasone-treated sickle mice and 19% of the venules became static in the placebo-treated sickle mice (P < 0.001, placebo vs. dexamethasone at 24 h). After exposure of the sickle mice to a second round of hypoxia-reoxygenation at 72 h posttreatment, the percentage of static venules in dexamethasone-treated sickle mice increased to 60%, whereas in the placebo-treated mice, 12% of the venules were static (P < 0.001, placebo vs. dexamethasone at 72 h). Thus dexamethasone-treated mice, but not placebo-treated mice, had a significant increase, or rebound, in stasis 72 h after dexamethasone withdrawal.
To determine whether the rebound in stasis was associated with an increase in adhesion molecule expression, we measured VCAM-1 and ICAM-1 expression after hypoxia-reoxygenation in the livers, lungs, and skin at 24 and 72 h after dexamethasone withdrawal (Fig. 8, A and B). VCAM-1 expression (Fig. 8A) in the liver nearly doubled at 72 h postdexamethasone compared with that at 24 h (P < 0.16, 24 vs. 72 h). In the lungs, VCAM-1 expression (Fig. 8A) significantly increased from 69% of placebo-treated sickle mice at 24 h to 111% at 72 h postdexamethasone (P < 0.05, 24 vs. 72 h). In the dorsal skin, VCAM-1 expression (Fig. 8A) in dexamethasone-treated sickle mice increased more than threefold from 24 to 72 h postdexamethasone (P < 0.001, 24 vs. 72 h). Thus the lungs and skin had significant increases in VCAM-1 expression 72 h after dexamethasone withdrawal (Fig. 8A) compared with significant reductions in VCAM-1 expression in the same organs 24 h after dexamethasone withdrawal.
ICAM-1 expression (Fig. 8B) in the liver did not significantly increase at 72 h compared with 24 h postdexamethasone (P < 0.13, 24 vs. 72 h). However, ICAM-1 expression (Fig. 8B) in the lungs of dexamethasone-treated sickle mice doubled at 72 h postdexamethasone (P < 0.001, 24 vs. 72 h). ICAM-1 expression (Fig. 8B) in the skin of dexamethasone-treated sickle mice also increased significantly at 72 h postdexamethasone (P < 0.05, 24 vs. 72 h). Thus the lungs and skin had significant increases in ICAM-1 expression 72 h after dexamethasone withdrawal (Fig. 8B) compared with significant reductions in ICAM-1 expression in the same organs 24 h after dexamethasone withdrawal.
Considerable evidence has accrued that sickle cell disease is accompanied by a robust inflammatory response. Not only do vasoocclusive crises occur during inflammatory insults such as infections or surgery, but the steady-state patient has elevated leukocyte counts (3, 55), activated leukocytes (7, 21, 25, 33, 59) and platelets (1, 39, 56), elevated cytokines (14, 23, 34), and circulating endothelial cell adhesion molecules (13). Vasoocclusion in sickle cell disease is transient and episodic (18, 29), which would promote tissue ischemia and reperfusion. Reperfusion of tissues after interruption of their vascular oxygen supply causes free radical generation that leads to tissue damage, a scenario referred to as “reperfusion injury.” Support for this ischemia-reperfusion model of vasoocclusion comes from the observation that transgenic sickle mice generate excessive ROS after a transient induction of enhanced RBC sickling by systemic hypoxia (31, 38). Kaul and Hebbel (31) showed that such oxidative stress-induced ROS in sickle mice were the cause of an inflammatory response that included leukocyte rolling and adhesion as well as impaired venular blood flow. Also, xanthine oxidase, an enzyme that is activated by tissue hypoxia and that produces superoxide radicals and hydrogen peroxide during reperfusion, is significantly elevated in the plasma of human sickle patients and in the lungs and plasma of transgenic sickle mice (2, 42). These findings support the concept of ischemia-reperfusion physiology helping to promote the inflammatory phenotype seen in sickle cell patients and in transgenic sickle mice.
The inflammatory response to hypoxia can be blunted by anti-inflammatory therapy, antioxidant treatment, and nitric oxide administration (32, 57, 58). Thus the potential utility of anti-inflammatory agents in interrupting these processes seems reasonable. Glucocorticosteroids have been the mainstay for treating inflammatory disorders for nearly 70 yr. Thus it is not surprising that glucocorticosteroids have been used to ameliorate symptoms in sickle cell disease. High doses have reduced the duration of hospitalization in children with painful crises and modulated severe acute chest syndrome (8, 24). Because of side effects, immunosuppressive impact, and rebound attacks, glucocorticosteroids have not been widely used in patients with sickle cell disease. Because glucocorticosteroids can inhibit NF-κB activation and VCAM-1 and ICAM-1 expression, among their many anti-inflammatory actions, we wondered whether inhibition of endothelial cell activation underlies their beneficial effects in sickle cell disease.
In the studies presented, dexamethasone prevented decreases in blood flow in the subcutaneous venules of sickle mice after hypoxia-reoxygenation (Table 1). Transient vasoocclusion was observed in the subcutaneous venules of placebo-treated sickle mice after exposure to hypoxia-reoxygenation (Table 2). Dexamethasone-treated sickle mice and untreated normal control mice did not develop stasis in their subcutaneous venules after hypoxia-reoxygenation (Table 2). Dexamethasone treatment also inhibited leukocyte adhesion induced by hypoxia-reoxygenation in the venules of sickle mice (Table 3). Vasoocclusion in sickle mice is associated with the presence of clumps of adherent leukocytes at bifurcations near the beginning of static venules (29). These adherent leukocytes appear to plug the static venules (29). Recent intravital microscopy studies by Turhan and colleagues (50, 51) in sickle mice suggest that adherent leukocytes in postcapillary venules may play a critical role in vasoocclusion by capturing circulating sickle RBCs. The investigators discovered that commercial intravenous human immune globulin preparations at concentrations of 200 mg/kg inhibit RBC-leukocyte interactions in cremasteric venules and improve blood flow and survival in a dose-dependent manner. In our present study, VCAM-1 or ICAM-1 blockade at doses of only 30 μg of MAb per mouse, or ∼1.2 mg/kg, prevented stasis and leukocyte adhesion, but not rolling, in the subcutaneous venules of sickle mice after hypoxia-reoxygenation (Table 4 and Fig. 1). Although previously it was shown that P-selectin blockade or knockout improves murine sickle blood flow after an inflammatory response was induced (19, 31, 51), this is the first demonstration that VCAM-1 or ICAM-1 blockade inhibits hypoxia-induced vasoocclusion. This finding is of interest because it reveals an important molecular insight into the pathobiology of vasoocclusion in sickle cell disease. Our studies demonstrating blockade of leukocyte adhesion, but not of rolling, by anti-VCAM-1 and anti-ICAM-1 MAbs suggests that the vasculature requires adhesion of blood cells (RBC/leukocytes) to the endothelium for stasis to occur. Of relevance to this observation, a recent study (13) demonstrated that the sickle crises-inhibiting drug hydroxyurea decreased circulating VCAM-1 and ICAM-1 in sickle cell patients, suggesting another potential mechanism through which hydroxyurea could be inhibiting vasoocclusive crises.
Despite studies that have now linked P-selectin, VCAM-1, and ICAM-1 to vasoocclusion in transgenic sickle mice, the relative contributions of sickle RBCs and leukocytes to the vasoocclusive process remain unclear. Although leukocyte binding to these adhesion molecules has been well known for many years, more recent studies also have revealed binding of sickle RBCs to P-selectin and VCAM-1 (19, 35, 43). Additional studies are needed to dissect the relative contributions of sickle RBCs and leukocytes to vasoocclusion. Regardless of the outcome of these studies, the ultimate trigger to the vasoocclusive process must be related to the mutation in the sickle β-globin gene in the RBC.
Our data demonstrate an association among endothelial cell activation, adhesion molecule expression, and vasoocclusion. NF-κB was activated in the liver, lungs, and skin of transgenic sickle mice and was further increased in the livers and skin, but not the lungs, by hypoxia-reoxygenation (Fig. 2). Dexamethasone treatment reduced NF-κB, VCAM-1, and ICAM-1 in the liver, lungs, and skin of sickle mice after hypoxia-reoxygenation (Figs. 3–6). ICAM-1 expression in the liver was the only marker of endothelial cell activation that was resistant to dexamethasone treatment (Fig. 6). This finding suggests that the heterogeneity of responses to dexamethasone reflect organ specificity and endothelial cell diversity. ICAM-1 can be expressed on cells other than endothelial cells such as pneumocytes (54) or hepatocytes (52) that may not be directly affected by the flow of sickle RBC in the vasculature. Furthermore, expression of these adhesion molecules depends on other transcription factors as well as synthesis and degradation rates. Thus this differential tissue response of ICAM-1 to dexamethasone is not surprising.
Stasis in the skin (Fig. 7) and VCAM-1 and ICAM-1 expression in the lungs and skin (Fig. 8) rebounded significantly 72 h after dexamethasone withdrawal. The mechanism of the rebound may be due to an overshoot of expression of inflammatory adhesion molecules upon the “release” of dexamethasone suppression. For example, short-term use of dexamethasone can temporarily suppress radiation induced proinflammatory cytokine gene expression in mouse lungs, but there may be a strong rebound effect after drug withdrawal that lasts up to 3 days (27). Markers of inflammation in the skin of sickle mice mirrored the inflammation seen in other metabolically active organs. These observations suggest that the inflammation and vasoocclusion seen in the skin of sickle mice can be generalized to other organs. Taken together, these data suggest that vasoocclusion is linked to endothelial cell activation and adhesion molecule expression.
This work is novel in several important ways compared with previous work. First, vascular stasis is linked to NF-κB activation and to VCAM-1 and ICAM-1 expression for the first time. Previous work by Frenette’s laboratory (51) demonstrated a direct link between P-selectin and stasis; this is the first time that VCAM-1 and ICAM-1 have been directly linked to stasis. Second, rebounds in stasis after dexamethasone withdrawal are linked to rebounds in VCAM-1 and ICAM-1 expression. This study, for the first time, provides a molecular rationale for previously observed rebounds in vasoocclusive pain crises after glucocorticoid withdrawal in sickle cell disease patients. In our study, we equate stasis with vasoocclusion for experimental purposes, but we do not necessarily equate the two clinically. Stasis is transient, whereas vasoocclusion may be longer lasting and may involve coagulation and fibrin deposition, leading to organ infarction and injury. Remarkably, our studies in the S+S-Antilles model show that vascular stasis, induced by hypoxia-reoxygenation, is indeed a rapid process of relatively short duration; stasis occurs in the subcutaneous venules within 1 h of reoxygenation and resolves in ∼50% of the venules by 4 h and completely within 24 h (29). We do not know if vasoocclusion resolves completely without intervention after 24 h in human sickle cell disease patients as it does in our murine model. Vasoocclusive pain crises in human sickle patients tend to last several days, suggesting that resolution of vasoocclusion in human sickle patients may take longer than 24 h.
These findings provide support for our hypothesis that anti-inflammatory agents may modulate vasoocclusion in sickle cell disease. We speculate that glucocorticosteroids may improve symptoms and modulate the acute chest syndrome by inhibiting NF-κB activation, thereby decreasing adhesion molecules VCAM-1 and ICAM-1 and promoting blood flow. Caution regarding the use of dexamethasone for treatment of sickle cell disease patients must be heeded, because steroids can compromise host defense mechanisms, and patients treated with glucocorticosteroids often have rebound attacks (8, 24). These data suggest that these rebound attacks are caused by rebounds in endothelial cell activation and vascular stasis after withdrawal of glucocorticosteroids. Our hope is that more long-lasting, specific, and targeted agents such as anti-VCAM-1 and anti-ICAM-1 therapy or antioxidants will be developed to decrease endothelial cell activation and vascular inflammation while maintaining host defenses. Further studies are certainly warranted to test anti-inflammatory agents that may provide new and novel therapies for sickle cell disease.
This work was supported by National Heart, Lung, and Blood Institute Grants HL-67367 and HL-55552.
We thank Tyson Rogers for insightful comments and help with the statistical analysis.
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- Copyright © 2005 by the American Physiological Society