Alpha4-integrin-VCAM-1 binding mediates G protein-independent capture of encephalitogenic T cell blasts to CNS white matter microvessels

Abstract

Direct in vivo evidence is still lacking for alpha4-integrin-mediated T cell interaction with VCAM-1 on blood-brain barrier-endothelium in experimental autoimmune encephalomyelitis (EAE). To investigate a possible alpha4-integrin-mediated interaction of encephalitogenic T cell blasts with VCAM-1 on the blood-brain barrier white matter endothelium in vivo, we have developed a novel spinal cord window preparation that enabled us to directly visualize CNS white matter microcirculation by intravital fluorescence videomicroscopy. Our study provides the first in vivo evidence that encephalitogenic T cell blasts interact with the spinal cord white matter microvasculature without rolling and that alpha4-integrin mediates the G protein-independent capture and subsequently the G protein-dependent adhesion strengthening of T cell blasts to microvascular VCAM-1.

Figure 1

Spinal window preparation for direct intravital microscopic assessment of white matter microcirculation. Stereomicroscopic (a) and intravital fluorescence microscopic (b) view of spinal window preparation exposing the dorsal spinal cord. Bar, 500 μm. High-magnification intravital fluorescence microscopy of spinal microvasculature depicting white matter capillary network (arrows) (c) and white matter postcapillary venules draining into the central dorsal vein (d). Bar, 100 μm.

Figure 2

T lymphoblast/endothelium interaction within spinal white matter postcapillary venules during cell infusion. (a) Postcapillary venular segment before cell infusion after contrast enhancement of spinal microvasculature using FITC-dextran₁₅₀. Arrows indicate direction of microvascular blood flow. (b–e) Intravital microscopic sequence of two Cell Tracker Orange–labeled T lymphocytes (1 and 2) over 0.24 seconds within the identical postcapillary segments indicated in a. Cells either lacked interaction with endothelium or were captured to endothelium without prior rolling. Bar, 100 μm.

Figure 3

Normalized velocity of T cell blasts. Objective assessment of T lymphoblast/endothelial interaction was obtained by comparing the velocity distribution of T cells observed in vessels of comparable size. For the control group, 198 cells in eight venules of two mice, for the anti–α4-group, 440 cells in 22 venules of four mice, and for the anti–VCAM-1 group 236 cells in 15 venules of three mice were analyzed. V_crit, the velocity of an idealized noninteracting T cell blast, was calculated as described in Methods. Five percent of circulating T cells were transiently captured at the vascular wall (a, c). Blocking α4-integrin (a) or VCAM-1 (c) resulted in a significantly reduced number of captured T cells (a, c). Lack of T lymphoblast rolling is demonstrated by the lack of T cells traveling at velocities below V_crit (b, d).

Figure 4

Involvement of α4-integrin and VCAM-1 in permanent T lymphoblast adherence within spinal cord white matter microvasculature. (a–d) Permanent T lymphoblast adherence in control mice 10 minutes after cell injection. T cell blasts were permanently adherent either within the capillary network (a and b) or within postcapillary venules (c and d). Intravital fluorescence videomicroscopy using epi-illumination techniques. Contrast enhancement of spinal microvasculature using FITC-dextran₁₅₀ (a and c; arrows mark localization of T cells). Cell Tracker Orange–labeled T lymphocytes (b and d). Bar, 100 μm. (e–j) Permanent T lymphoblast adherence 1 hour after cell injection. Control (e and f), anti–α4-integrin Ab (g and h), anti–VCAM-1 (i and j). Intravital fluorescence videomicroscopy using epi-illumination techniques. Contrast enhancement of spinal microvasculature using FITC-dextran₁₅₀ (e, g, and i; arrows mark localization of T cells). Cell Tracker Orange–labeled T lymphocytes (f, h, and j). Bar, 100 μm. (k) Quantitative analysis of permanent T lymphoblast adherence. T lymphoblasts permanently adhering within spinal cord white matter microvasculature were counted 10 minutes, 1 hour, and 2 hours after infusion of 3 × 10⁶ PLP-specific T cell blasts by intravital fluorescence videomicroscopy using epi-illumination techniques as described in Methods. Number of mice included in this analysis per group: control, n = 5 mice; antibody-control, n = 2 mice; anti–α4-integrin group, n = 3 mice; and anti–VCAM-1 group, n = 3 mice. Asterisks indicate significant differences. p.i., postinjection.

Figure 5

Requirement for G proteins in T lymphoblast interaction with the spinal cord white matter microvasculature. (a) Presence of high-affinity α4-integrin on encephalitogenic T cell blasts but not on resting T cells is demonstrated by binding of VCAM-1–Ig (thick line) as determined by FACS-analysis. Binding of HT7-Ig was used as control (thin line). (b and c) Capture events (b) and normalized velocity (c) of pertussis toxin–pretreated T cell blasts. Objective assessment of PTX-pretreated T lymphoblast/endothelial interaction was obtained by comparing the velocity distribution of T cells observed in vessels of comparable size. For the control group 160 cells in seven postcapillary venules of two mice and for the pertussis toxin group 190 cells in 15 postcapillary venules of three mice were analyzed. V_crit, the velocity of an idealized noninteracting T cell blast, was calculated as described in Methods. Ten percent of circulating T cells were transiently captured at the vascular wall (b). PTX did not influence the number of captured T cells (b). Lack of T lymphoblast rolling is demonstrated by the lack of T cells traveling at velocities below V_crit (c). (d) Quantitative analysis of permanent T lymphoblast adherence within spinal cord white matter microvasculature. T lymphoblasts permanently adhering within spinal cord white matter microvasculature were counted 10 minutes, 1 hour, and 2 hours after infusion of 3 × 10⁶ PLP-specific T cell blasts by intravital fluorescence videomicroscopy using epi-illumination techniques as described in Methods. Number of mice included in this analysis per group: PBS-control, n = 2; MTX:, n = 2; PTX, n = 3. Asterisks indicate significant differences.

Figure 6

Localization of T lymphoblasts within the spinal cord parenchyma in control animals. Six hours after infusion of T lymphoblasts, the Cell Tracker Orange–labeled T cell blasts (red fluorescence) could be localized outside the spinal cord microvasculature (green fluorescence) within the spinal cord parenchyma of control animals. Superimposed fluorescence is shown, which demonstrates one T cell blast attached within the venule (yellow fluorescence) and one T cell blast within the spinal cord parenchyma (red fluorescence). Bar, 10 μm.