Human spinal cord injury causes specific increases in surface expression of β integrins on leukocytes

Abstract

Spinal cord injury (SCI) activates circulating leukocytes that migrate into the injured cord and bystander organs using adhesion molecule-mediated mechanisms. These cells cause oxidative damage, resulting in secondary injury to the spinal cord, as well as injury to bystander organs. This study was designed to examine, over a 6-h to 2-week period, changes in adhesion molecule surface expression on human peripheral leukocytes after SCI (9 subjects), using as controls 10 uninjured subjects and 6 general trauma patients (trauma controls, TC). Both the percentage of cells expressing a given adhesion molecule and the average level of its expression was quantified for both circulating neutrophils and monocytes. The percentage of neutrophils and monocytes expressing the selectin CD62L was unchanged in TC and SCI patients after injury compared to uninjured subjects. Concurrently, the amount of surface CD62L on neutrophils was decreased in SCI and TC subjects, and on monocytes after SCI. The percentage of neutrophils expressing α4 decreased in TC, but not in SCI, subjects. Likewise, the percentage of neutrophils and monocytes expressing CD11d decreased markedly in TC subjects, but not after SCI. In contrast, the mean surface expression of α4 and CD11d by neutrophils and monocytes increased after SCI compared with uninjured and TC subjects. The percentage of cells and surface expression of CD11b were similar in neutrophils of all three groups, whereas CD11b surface expression increased after SCI in monocytes. In summary, unlike changes found after general trauma, the proinflammatory stimulation induced by SCI increases the surface expression of adhesion molecules on circulating neutrophils and monocytes before they infiltrate the injured spinal cord and multiple organs of patients. Integrins may be excellent targets for anti-inflammatory treatment after human SCI.

FIG. 1

An example of flow cytometry analysis of surface protein (CD62L) expression in neutrophils and monocytes. Expression was detected by fluorescent antibodies to the surface antigens. Neutrophils and monocytes were gated by their characteristic forward- and side-scatter profiles (A), and then cells in each respective gate (B) were analyzed using two methods for mean fluorescence intensity (MFI). First, fluorescence was plotted against side-scatter to obtain the percentage of cells expressing the protein (C and E). Background fluorescence generated by the isotype-matched control antibody (left quadrants) was used to define the gating for detection of cells expressing the protein (right quadrants). Next, fluorescence was plotted against cell number and the area under this histogram was used as MFI (D and F). Again, fluorescence of the isotype-matched antibody was used to determine background fluorescence. Normalized MFI of each surface protein was calculated by subtracting the area of background fluorescence from the area of fluorescence of the specific protein.

FIG. 2

The L-selectin (CD62L) expression on neutrophils and monocytes of uninjured (U), trauma control (TC), and spinal cord injury (SCI) subjects. Panels A and B illustrate examples of fluorescence plotted against side-scatter to obtain percentages of neutrophils (A) and monocytes (B) expressing CD62L. Gating was defined as shown by the fluorescence generated after incubation with the isotype-matched control antibody (Isotype). The larger numerals in one of each of the quadrants indicate the percentage of positive cells in that quadrant. The percentages of neutrophils and monocytes expressing CD62L were similar in these examples from an uninjured subject (Uninjured), and from a TC and a SCI subject, obtained 24 h after injury. Averages and typical examples of mean fluorescence intensity (MFI) in neutrophils and monocytes from the uninjured group, and from the TC and SCI groups, plotted at 6–72 h, 1 week, and 2 weeks after injury, are shown in panels C and D. The left panels illustrate mean values from each subject group, plotted at the indicated sampling times. The right panels show a typical histogram example for each group, including the isotype control. TC and SCI examples are for 24 h post-injury. Neutrophils from TC and SCI subjects had decreased CD62L MFI (mean ± standard error) compared to that of uninjured subjects. Decreases in monocytes were significant at all time points after SCI, but only at 6 h and 2 weeks in the TC subjects (*p ≤0.05 versus the uninjured group, n =5). Animal numbers at 6, 12, 24, 48, and 72 h, and 1 and 2 weeks after injury, respectively: TC: n =4, 5, 5, 5, 4, 3, 3; SCI: n =3, 6, 6, 6, 4, 5, 3.

FIG. 3

The α4 subunit expression on neutrophils and monocytes of uninjured (U), trauma controls (TC), and spinal cord injury (SCI) subjects. Panels A and B illustrate examples of fluorescence plotted against side-scatter to obtain percentages of neutrophils (A) and monocytes (B) expressing α4. The format of this figure is identical to that of Figure 2. The percentages of neutrophils expressing α4 in this example at 24 h after injury were lower in the TC than in the uninjured subjects, whereas the SCI subjects were similar to the uninjured subjects. The percentages of monocytes expressing α4 in the uninjured, TC, and SCI subjects were similar. Mean fluorescence intensity (MFI) (±standard error) for α4 on neutrophils and monocytes was decreased in TC subjects compared to uninjured and SCI subjects, at both early and late sampling times (C and D). In contrast, after SCI, α4 MFI on neutrophils was increased compared to uninjured and TC subjects. α4 MFI on monocytes was greater in SCI than TC subjects (*p ≤0.05 versus uninjured subjects; #p ≤0.05 versus TC subjects; U: n =6 at all time points). Animal numbers at 6, 12, 24, 48, and 72 h, and 1 and 2 weeks after injury, respectively: TC: n =5, 6, 6, 6, 5, 4, 4; SCI: n =4, 8, 8, 8, 5, 6, 4.

FIG. 4

CD11d subunit expression on neutrophils and monocytes of uninjured (U), trauma controls (TC), and spinal cord injury (SCI) subjects. Panels A and B illustrate examples of fluorescence plotted against side-scatter to obtain percentages of neutrophils (A) and monocytes (B) expressing CD11d. The format of this figure is identical to that of Figure 2. The percentages of neutrophils expressing CD11d in this example at 48 h after injury were lower in the TC than in the uninjured subjects, and were higher in the SCI subjects. The percentages of monocytes expressing CD11d in the TC subjects was lower than that in the uninjured subjects, but in the SCI subjects this percentage was similar to that of the uninjured subjects. Mean fluorescence intensity (MFI) (± standard error) is shown in panels C and D. The histogram examples were taken at 48 h after injury. CD11d MFI was not changed in neutrophils or monocytes of TC subjects compared to uninjured subjects. After SCI, the CD11d MFI of neutrophils and monocytes was significantly greater than that of uninjured subjects and TC both at early and late sampling times (*p ≤0.05 versus uninjured subjects; #p ≤0.05 versus TC subjects; U: n =5). Animal numbers at 6, 12, 24, 48, and 72 h, and 1 and 2 weeks after injury: TC: n =5, 6, 6, 6, 5, 4, 4; SCI: n =4, 8, 8, 8, 5, 6, 4.

FIG. 5

CD11b subunit expression on neutrophils and monocytes of uninjured (U), trauma controls (TC), and spinal cord injury (SCI) subjects. Panels A and B illustrate examples of fluorescence plotted against side-scatter to obtain percentages of neutrophils (A) and monocytes (B) expressing CD11b. The format of this figure is identical to that of Figure 2. The percentages of neutrophils and monocytes expressing CD11b in these TC and SCI examples at 24 h after injury were no different from those in the uninjured subjects. Mean fluorescence intensity (MFI) (±standard error) for CD11b on neutrophils and monocytes was unchanged in TC subjects compared to uninjured subjects (C and D). CD11b MFI of neutrophils in SCI subjects was greater than that in uninjured and TC subjects only at 1 week after injury. In monocytes, the CD11b MFI was greater than that of uninjured and TC subjects at several early and later sampling times (*p ≤ 0.05 versus uninjured subjects; #p ≤ 0.05 versus TC subjects; U: n =5). Animal numbers at 6, 12, 24, 48, and 72 h, and 1 and 2 weeks after injury, respectively: TC: n =5, 6, 6, 6, 5, 4, 4; SCI: n =4, 7, 7, 7, 4, 5, 4.