Hypoxia/reoxygenation causes inflammatory response in transgenic sickle mice but not in normal mice

Abstract

In sickle cell anemia, the initiation, progression, and resolution of a vasoocclusive episode may present features of ischemia-reperfusion injury, with recurrent episodes of ischemia/hypoxia and reoxygenation promoting inflammation. Here, we have tested the hypothesis that hypoxia/reoxygenation triggers inflammation in the transgenic sickle mouse. In these mice, even at ambient air, peripheral leukocyte counts are elevated by 1.7-fold and neutrophil counts by almost 3-fold. Two hours of hypoxia, followed by reoxygenation, induced a greater than normal rolling flux and adhesion of leukocytes in these mice, but no leukocyte extravasation. When 3 hours of hypoxia was followed by reoxygenation, sickle mice, but not normal mice, showed a distinct inflammatory response characterized by an increased number of adherent and emigrated leukocytes. Because these events, which are exaggerated in sickle mice, are not seen in response to hypoxia alone, we conclude that they represent a form of reperfusion injury. Studies using an H(2)O(2)-sensitive probe revealed clear evidence of oxidant production in vascular endothelial cells after hypoxia/reoxygenation in sickle mice. Infusion of an anti-P-selectin antibody, but not an anti-E-selectin antibody, completely inhibited this inflammatory response and significantly increased wall shear rates. These findings suggest that leukocyte-endothelium interaction contribute to vasoocclusive events in the sickle mice and perhaps in human sickle disease.

Figure 1

Representative fluorographs of cremaster venules in β^S mice (top) and their corresponding DHR fluorescence profile analysis (bottom). (a) Normoxic control. (b) After 3 hours of hypoxia and 30 minutes of reoxygenation, the peaks of DHR activity correspond to fluorescence intensity in the vascular endothelial cells. Bar = 10 μm.

Figure 2

Peripheral leukocyte and neutrophil counts in control and β^S mice. ^AP < 0.005–0.003.

Figure 3

The effect of hypoxia/reoxygenation on leukocyte rolling velocity in control and β^S mice. ^AP < 0.001 versus corresponding wild-type controls (Student’s t test); ^BP < 0.05 versus respective normoxic values (Newman-Keuls multiple comparisons).

Figure 4

(a) The effect of hypoxia/reoxygenation on leukocyte rolling flux in control and β^S mice. (b) The effect of hypoxia/reoxygenation on leukocyte adhesion in control and β^S mice. ^AP < 0.01–0.0001 versus corresponding wild-type controls (Wilcoxon two-sample test); ^BP < 0.05 versus respective normoxic values (Kruskal-Wallis test for ANOVA).

Figure 5

Photomicrographs of cremaster venules after 3-hour hypoxia plus 18 hours of reoxygenation. (a) Normal mouse shows few emigrated leukocytes (arrowheads) in the cremaster. (b) In contrast, β^S mouse shows a large number of emigrated leukocytes adjacent to a venule (arrowheads). Large arrows depict the flow direction. Next to the venule in the upper middle part is the image of photodiode fibers for V_rbc measurement. Bar = 20 μm.

Figure 6

The effect of hypoxia/reoxygenation on leukocyte emigration in control and β^S mice. ^AP < 0.01 and ^BP < 0.0001 versus corresponding wild-type controls (Wilcoxon two-sample test).

Figure 7

(a) Hypoxia/reoxygenation in the β^S mice: the effect of anti–P- and anti–E-selectin antibodies on leukocyte rolling flux. (b) Hypoxia/reoxygenation in the β^S mice: the effect of anti–P- and anti–E-selectin antibodies on leukocyte adhesion. ^AP < 0.05 versus normoxic values; ^BP < 0.05 versus other groups (Newman-Keuls multiple comparisons).

Figure 8

Differences in dihydrorhodamine 123 (DHR) intensity (ΔI) between background and venular endothelial cells in normal and β^S mice during normoxia and after hypoxia/reoxygenation. β^S mice showed greater ΔI compared with corresponding wild-type controls in any experimental group (^AP < 0.0002–0.0001), with maximal ΔI recorded at 30 minutes after the hypoxic period. ^BP < 0.05 compared with respective normoxic values (Newman-Keuls multiple comparisons).