α4-integrin-VCAM-1 binding mediates G protein–independent capture of encephalitogenic T cell blasts to CNS white matter microvessels (original) (raw)

Mice. Female SJL/N mice were obtained from Bomholdgård Breeding (Ry, Denmark) and used for experiments at the age of 17 to 19 weeks.

Pertussis toxins and mAb’s. The rat anti-mouse mAb’s used in this study were PS/2 (anti–α4-integrin rat IgG2b), 6C7.1 (anti–VCAM-1 rat IgG1), and MJ7/18 (anti-endoglin IgG2a), and they have been described in great detail previously (2, 5). The mAb’s were purified from serum-free hybridoma supernatants, and endotoxin levels were determined by Fresenius (Taunusstein, Germany) were below detection levels (< 0.6 EU/ml). Pertussis toxin (PTX) was obtained from Sigma-Aldrich Chemie GmbH (Deisenhofen, Germany). Mutant PTX (MTX; PT9K/129G) lacking the enzymatic activity of PTX was used as a control. MTX was kindly provided by R. Rappuoli (Chiron SpA., Siena, Italy) (13).

T lymphocyte lines and induction of EAE. Establishment and culture of the CD4+ MHC class II–restricted protein lipid protein-specific (PLP-specific) T cell lines derived from SJL/N mice and induction and treatment of EAE have all been described in great detail previously (2, 4).

Labeling of T lymphoblasts. T cell blasts were labeled with 20 nM Cell Tracker Orange (Molecular Probes, Eugene, Oregan, USA) in RPMI-1640 with 10% FCS at 5 × 106 cells/ml and 37°C for 45 minutes, according to the protocol by Hamann and Jonas (14). Cell Tracker Orange labeling did not impair encephalitogenicity of PLP-specific T cell blasts, because transfer of 3 × 106 PLP-specific Cell Tracker Orange–labeled T cell blasts into syngeneic animals induced EAE with the same time, kinetic, and clinical severity as the transfer of unlabeled PLP-specific T cell blasts when compared in six mice per group in two different experiments.

Flow cytometry. Flow cytometry using Ig chimeras was performed exactly as described previously (4). Human recombinant VCAM-1–IgG (kindly provided by Dirk Seiffge, Aventis Pharma, Frankfurt, Germany) or murine HT7-IgG (4) used as negative control IgG-fusion protein was used at 2.5 μg/ml.

Preparation of the spinal cord window. The surgical preparations and experiments were performed in accordance with the German legislation on the protection of animals and the Guide for the Care and Use of Laboratory Animals. Animals were anesthetized by subcutaneous injection of ketamine/xylazine. For systemic administration of fluorescent markers and injection of cells, a polyethylene catheter (PE-10) was inserted into the right common carotid artery of a thermocontrolled mouse placed on a heating pad. Next, the animal was turned to the prone position, and the head was fixed in a stereotactic rodent head holder. After a midline skin incision of 3–4 cm, the paravertebral musculature was detached from the cervical spinous processes and retracted laterally, exposing the vertebral laminae. Using microsurgical techniques, a laminectomy of C1 to C7 was performed, and the dura was opened over the dorsal spinal cord avoiding trauma to the parenchyma and the spinal microvasculature. To prevent dehydration and the influence of the ambient oxygen, the site was covered with an impermeable transparent membrane. Mice that were traumatized during surgery or revealed signs of acute inflammation (distorted vessels, hyperemia, stagnant blood flow) were excluded from the experiments.

Intravital fluorescence videomicroscopy. For intravital fluorescence videomicroscopy the animals were transferred to the microscope stage, remaining within the stereotactic head holder. Intravital fluorescence videomicroscopy was performed by epi-illumination techniques using a modified Axiotech Vario microscope with a 100-W mercury lamp emitting an adjustable light intensity (Attoarc; Carl Zeiss Jena GmbH, Jena, Germany), which was attached to a combined blue (450–490 nm) and green (520–570 nm) filter block (Carl Zeiss Jena GmbH). Observations were made using ×3.2 long-distance, ×10 long-distance, and ×20 water immersion working objectives (all from Carl Zeiss Jena GmbH), resulting in magnifications of ×50, ×200, and ×400, respectively. Microscopic images were recorded by means of a low-light level charged coupled device video camera with an optional image intensifier for weak fluorescence (Kappa Opto-Electronics GmbH, Gleichen, Germany) and were transferred to a S-VHS videosystem (Panasonic, Hamburg, Germany) for off-line evaluation.

The spinal cord microvasculature was visualized by contrast enhancement with 2% FITC-conjugated dextran (0.1 ml FITC-dextran150, intravenously; MW = 150,000; Sigma Chemical Co., St. Louis, Missouri, USA) and use of the blue-light epi-illumination. Simultaneously, injected Cell Tracker Orange–labeled T cell blasts were visualized within the spinal cord microcirculation using the green-light epi-illumination. This combination of two fluorescent markers with distinct excitation wavelengths allowed localization of Cell Tracker Orange–labeled T cell blasts with respect to FITC-stained vessel lumina.

Experimental protocol. Following visualization of the spinal cord microvasculature by FITC-dextran150, 3 × 106 Cell Tracker Orange–labeled PLP-specific T cell blasts were injected in aliquots of 100 μl, each containing 106 T cells, and their interaction with the endothelium was assessed over an observation period of 60 seconds within the spinal capillary bed and postcapillary venules (20–60 μm) of three different microvascular regions of interest. In contrast, due to the angioarchitecture of the spinal microvasculature, T cell blast/endothelium interaction within precapillary arterioles, which are located at the depth of the spinal cord parenchyma, could not be visualized by intravital fluorescence videomicroscopy. To assess permanent T cell blast adhesion, the spinal cord microvasculature was scanned at 10 minutes, 1 hour, and 2 hours after cell injection.

To study the role of α4-integrin and VCAM-1 for T cell blast/endothelial interaction, 3 × 106 T cell blasts were preincubated with 90 μg PS/2 in 300 μl PBS for 20 minutes (n = 3 animals) or mice were injected with 90 μg 6C7.1 in 150 μl PBS 20 minutes before infusion of T lymphoblasts (n = 3), respectively. Animals treated with PBS (n = 5) or the control mAb MJ7/18 (n = 2) served as controls. MJ7/18 was chosen since it binds to the vascular wall and has been shown previously to have no influence to the development of EAE (2). In separate experiments T lymphoblasts were incubated with PTX (n = 3) or MTX (n = 2) (each 100 ng/ml) for 2 hours at 37°C in order to study the relevance of G protein–mediated activation of α4-integrin (n = 3). Unbound PTX or MTX was removed by washing the T cells before injection into the mice.

Intravital microscopic image analysis. Quantitative analysis of the spinal cord microcirculation included the diameter of postcapillary venules (d) and the velocity of nonadherent T lymphoblasts. The highest cell velocity per venule, _V_max, was used to calculate the mean blood flow velocity, as described previously (15): _V_mean =_V_max /(2 – ε2) (μm/s) where ε is the ratio of the T lymphoblast diameter to vessel diameter (d). From _V_mean and d, wall shear rate (γ) was estimated as γ = 8 × _V_mean /d (s–1) (15), and the shear stress (τ) was approximated by τ = γ × 0.025 (dyn/cm2) (16).

In postcapillary venules permanently adherent T lymphoblasts were identified as cells that stuck to the vessel wall without moving or detaching from the endothelium within an observation period of at least 20 seconds. Nonpermanently adherent T lymphoblasts were further categorized using a velocity criterion derived from the assumption of a parabolic velocity profile in the microvessel (15). Therefore, _V_crit, the velocity of an idealized noninteracting cell traveling at the vessel wall, was calculated as _V_crit = _V_mean × ε × (2 – ε). Consequently, any cell traveling below _V_crit was regarded as a cell interacting with the vessel wall; any cell traveling above _V_crit was regarded as a noninteracting cell (15, 17). Within the capillary network, plugging T lymphoblasts were defined as cells that did not move and obviously blocked the capillary lumen, inducing blood flow stasis in the corresponding vascular segment. Permanent T lymphoblast adhesion at 10 minutes, 1 hour, and 2 hours after cell injection is expressed as the number of both adherent and plugging T lymphoblasts per region of interest (mm–2).

Detection of Cell Tracker Orange–labeled PLP-specific T lymphoblasts by immunofluorescence. Mice were perfused with 1% formaldehyde in PBS 2, 4, 6, and 8 hours after injection of the PLP-specific T cell blasts. Spinal cords were removed, embedded in Tissue-Tek (OCT; Miles Inc., Vogel, Giessen, Germany) and snap-frozen in a 2-methylbutane (Merck KGaA, Darmstadt, Germany) bath at –80°C. Serial longitudinal cryo-sections (6 μm) of the spinal cords were cut, air-dried overnight, and acetone fixed. Cell Tracker Orange–labeled T lymphoblasts were detected by immunofluorescence microscopy. Vessels on sections containing T lymphoblasts were visualized by staining with either the mAb MJ7/18, a rat anti–mouse endoglin, or with the mAb 9DB3, a rat anti–mouse VCAM-1, both at 10 μg/ml in PBS/0.1% BSA, followed by staining with a goat anti-rat–FITC IgG (10 μg/ml in PBS supplemented with 10% normal mouse serum; Jackson, Dianova GmbH, Hamburg, Germany). Alternatively, bound 6C7.1 or bound MJ7/18 Ab’s were detected by staining with a goat anti-rat–FITC (Jackson, Dianova GmbH). Sections were coverslipped with Mowiol 4-88 (Calbiochem-Novabiochem GmbH, Bad Soden, Germany) and immediately analyzed.

Statistics. Quantitative data are given as mean values plus or minus SD. Mean values were calculated from the average values in each animal. For analysis of differences between the groups, ANOVA followed by unpaired Student t test and Bonferroni correction for repeated measurements were performed. Results with P values less than 0.05 were considered significant and are marked with an asterisk.