CD44 is involved in selective leucocyte extravasation during inflammatory central nervous system disease

Abstract

Clinical signs of experimental autoimmune encephalomyelitis (EAE) are associated with the selective recruitment of CD4+ memory (CD45RBlow CD44high) T cells into the central nervous system (CNS). However, we have found that many of these recently recruited memory cells are CD44low, suggesting that the CD44 antigen may be involved in, and transiently lost during, the extravasation process. Indeed, administration of a CD44-specific antibody (IM7.8.1) induced leucocyte CD44 shedding and both prevented the development and ameliorated the severity of established EAE by inhibiting mononuclear cell infiltration into the CNS. Trafficking of cells into lymph nodes, however, a property mainly of naïve cells, was essentially unaffected. In contrast, treatment with antibody to very late activation antigen-4 (VLA-4) prevented homing to both the CNS and to lymph nodes. This study contests previous reports that dismissed a role for CD44 in inflammation of the CNS and, coupled with observations in murine dermatitis and arthritis, suggests that CD44 is involved in the homing of primed lymphocytes to sites of inflammation. CD44 should therefore be considered a target for immunotherapy of T-cell-mediated inflammatory diseases, such as multiple sclerosis.

Figure 1

Anti‐CD44 monoclonal antibody (mAb) IM7 induces the loss of CD44 from lymphocytes in vivo. Animals were injected intraperitoneally (i.p.), daily for 5 days, with 100 µg of either IM7 mAb or rat immunoglobulin. Inguinal lymph nodes were removed 12 hr after the last injection and the level of CD44 on CD4⁺ T cells was analysed by direct immunofluorescence using CD44‐specific KM201 mAb. The y‐axis shows cell number and the x‐axis is mean fluorescence intensity. Background staining gave an intensity of less than 10 fluorescence units.

Figure 2

Anti‐CD44 monoclonal antibody (mAb) IM7 inhibits the development and ameliorates the severity of established clinical disease. (a) Mice were injected with mouse spinal cord homogenate (SCH) in Freund’s adjuvant on days 0 and 7 and then injected intraperitoneally (i.p.), daily from day 12 postinjection (p.i.), with 0·1 ml of phosphate‐buffered saline (PBS) (untreated) (•), isotype rat immunoglobulin G2b (IgG2b) (YTS 169) control (▪) or CD44‐specific mAb IM7 (▴). (b) Animals were injected with SCH in Freund’s adjuvant on days 0 and 7. Following the onset of clinical disease on days 13–15 p.i., chronic‐relapsing experimental allergic encephalomyelitis (CREAE) mice were treated (▴) with five daily i.p. injections of 100 µg of anti‐CD44 mAb IM7 (•) or rat immunoglobulin control (▪). For both (a) and (b), the results represent the mean ± SEM clinical score of animals within each group following the start of mAb treatment.

Figure 3

Anti‐CD44 monoclonal antibody (mAb) IM7 inhibits lymphocyte entry into the central nervous system (CNS). Following the development of chronic‐relapsing experimental allergic encephalomyelitis (CREAE) 13–15 days after the injection of spinal cord homogenate (SCH) in Freund’s complete adjuvant, mice were given five daily injections of phosphate‐buffered saline (PBS) (a) or 100 µg of anti‐CD44 mAb IM7 (b). On day 18 postinjection (p.i.), spinal cords were removed, and sections were made and stained with haematoxylin and eosin. (a) A section from a control mouse with EAE containing large numbers of infiltrating cells. (b) A section from a corresponding area from a clinically well IM7‐treated mouse. Note the lack of infiltrating mononuclear cells.

Figure 4

In vitro shedding of CD44 prevents T‐cell migration into inflammatory central nervous system (CNS) lesions. Following the development of paralysis during acute‐phase chronic‐relapsing experimental allergic encephalomyelitis (CREAE), Biozzi AB/H (Thy1.1⁺) mice were injected intravenously (i.v.) with 1 × 10⁷ lymph node cells expressing Thy1.2 antigen, which had been treated with immobilized rat immunoglobulin or CD44‐specific IM7 monoclonal antibody (mAb) for 4 hr at 37°. Animals were perfused 6 hr later and single‐cell suspensions were prepared from the inguinal lymph nodes and spinal cord from each individual animal. Cells were gated to detect lymphocytes, and a dot‐plot was created showing double immunofluorescence flow cytometry of transferred Thy1.2⁺ T cells (fluorescence intensity on y‐axis) and the CD4⁺ T‐cell population (fluorescence intensity on x‐axis).