Antibodies to CD44 and integrin alpha4, but not L-selectin, prevent central nervous system inflammation and experimental encephalomyelitis by blocking secondary leukocyte recruitment

Abstract

The role of various adhesion molecules in lymphocyte homing to the brain and in inflammatory autoimmune disease of the central nervous system (CNS) was examined in mice. Activated T cell lines and clones expressed CD44 and integrin alpha4, but not L-selectin, and entered the CNS independent of their antigen specificity. mAbs directed against CD44 and integrin alpha4 prevented the transfer of experimental autoimmune encephalomyelitis (EAE) by myelin basic protein-specific T cells. T cells preincubated with anti-CD44 or antiintegrin alpha4 were blocked only partially from entering the brain parenchyma. However, both antibodies efficiently prevented CNS inflammation and clinical expression of EAE when injected in vivo. This effect lasted as long as antibodies were administered. Antibodies specific for L-selectin had no effect on homing of encephalitogenic T cells to the brain or development of EAE. Antiintegrin alpha4 and anti-CD44 did not impair the activation and function of encephalitogenic T cells in vitro and did not deplete integrin alpha4- or CD44-positive cells in vivo. These data suggest that, in the absence of leukocyte recruitment, the entry of a reduced number of activated myelin basic protein-reactive T cells in the CNS is not sufficient for the development and expression of EAE. We propose that antibodies to integrin alpha4 and CD44 prevent clinical disease by partially targeting the primary influx of encephalitogenic T cells and by preventing the secondary influx of leukocytes to lesions initiated by the transferred T cells.

Figure 1

Prevention of T cell-induced inflammation in the white matter of the CNS by antibodies directed against CD44 and integrin α₄ but not L-selectin. Mice were treated continuously with different antibodies for 8 days after adoptive transfer of 5 × 10⁶ MBP peptide Ac1-11-specific T cells. Thereafter, brains of mice treated i.p. with the indicated antibodies or PBS were removed, fixed, sectioned, and stained with hematoxylin and eosin. Typical regions were photographed. The original magnification was 200×. (a) Histopathology of mice treated with PBS. Shown is perivascular inflammatory infiltrate in the brainstem of a mouse with adoptive EAE. Clinical disease score at the time of death was grade 3. (b) Histopathology of mice treated with mAb MEL-14 (anti-L-selectin). Shown is perivascular inflammatory infiltrate in the brainstem of a mouse with adoptive EAE. Clinical disease score at the time of death was grade 2. (c) Histopathology of mice treated with mAb IM7.8.1 (anti-CD44). Shown is the absence of perivascular infiltrates in the brainstem of a mouse after adoptive transfer of encephalitogenic T cells. Clinical disease score was grade 0.

Figure 2

Prevention of adoptively transferred EAE by treatment with anti-CD44 and antiintegrin α₄ mAbs and amelioration of clinical signs by treatment with antiintegrin α_M mAbs. MBP peptide Ac1-11-specific T cells (5 × 10⁶/mouse, n = 5–15 mice/group) were transferred into naive (PL × SJL)F₁ mice after in vitro activation with antigen for 3 days. Mice in each group were treated twice (a) or over a longer time period (b) with mAbs IM7.8.1 (open circles), MEL-14 (closed circles), or PBS as control (open squares). Each mAb (250 μg) was given on the indicated days (marked by i). Mice were scored daily, and the mean EAE scores (± SEM) at each day after transfer are shown. The figure shows representative results from at least three experiments. In a second series of experiments (c and d), mice were treated with 250 μg of mAbs R1-2 (open circles), MEL-14 (closed circles), or PBS as control (open squares) on days as indicated by i. Mice were scored daily, and the mean EAE scores (± SEM) at each day after transfer are shown. The figures shows representative results from at least three experiments. (c) Continuous injections of antiintegrin α₄ mAb R1-2 prevent clinical signs of EAE as long as antibodies are administered. (d) During a mild course of EAE, continuous injections of R1-2 fully prevent clinical signs of EAE for at least 24 days. In the last series of experiments (e and f), mice were treated with antiintegrin α_M mAb M1/70 (e, full circles), 5C6 (f, full circles), or PBS as control (open squares). Clinical effects of treatment with these mAbs were moderate.

Figure 3

Effect of mAbs GK1.5 and IM7.8.1 on leukocyte subsets in vivo. Purified mAbs (250 μg) GK1.5, R1-2, IM7.8.1, or PBS were administered i.p. into naive (PL × SJL)F₁ mice (n = 5 per treatment group). Peripheral blood leukocytes were isolated 24 h later for counting and fluorescence-activated cell sorter analysis with directly conjugated reagents for the percentage of B cells (B220+), T cells (T200+), and CD4+ T cells (mAb GK1.5+). Results are expressed as mean ± SD of absolute cell counts (×1,000/μl of blood) adjusted to the total white blood cell counts as determined by the Coulter Counter.