Hyaluronan Anchored to Activated CD44 on Central Nervous System Vascular Endothelial Cells Promotes Lymphocyte Extravasation in Experimental Autoimmune Encephalomyelitis

Bruce F. Bebo,Michelle A. Berny-Lang,Steven G. Matsumoto,Asako Itakura,Owen J.T. McCarty,Scott C. Foster,Rubing Xing,Marnie A. Preston,Fatima Banine,Clayton W. Winkler,Larry S. Sherman

doi:10.1074/jbc.m112.356287

Bruce F. Bebo, Michelle A. Berny-Lang + Show 9 more

Open Access

https://doi.org/10.1074/jbc.m112.356287

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Abstract

The extravasation of lymphocytes across central nervous system (CNS) vascular endothelium is a key step in inflammatory demyelinating diseases including multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). The glycosaminoglycan hyaluronan (HA) and its receptor, CD44, have been implicated in this process but their precise roles are unclear. We find that CD44(-/-) mice have a delayed onset of EAE compared with wild type animals. Using an in vitro lymphocyte rolling assay, we find that fewer slow rolling (<1 μm/s) wild type (WT) activated lymphocytes interact with CD44(-/-) brain vascular endothelial cells (ECs) than with WT ECs. We also find that CD44(-/-) ECs fail to anchor HA to their surfaces, and that slow rolling lymphocyte interactions with WT ECs are inhibited when the ECs are treated with a pegylated form of the PH20 hyaluronidase (PEG-PH20). Subcutaneous injection of PEG-PH20 delays the onset of EAE symptoms by ~1 day and transiently ameliorates symptoms for 2 days following disease onset. These improved symptoms correspond histologically to degradation of HA in the lumen of CNS blood vessels, decreased demyelination, and impaired CD4(+) T-cell extravasation. Collectively these data suggest that HA tethered to CD44 on CNS ECs is critical for the extravasation of activated T cells into the CNS providing new insight into the mechanisms promoting inflammatory demyelinating disease.

Highlights

Multiple sclerosis (MS) is a demyelinating disease involving lymphocyte infiltration into the central nervous system (CNS)
We find that HA is tethered by CD44 to the luminal surface of TNF␣ stimulated endothelial cells (ECs) isolated from the brain and that slow rolling lymphocyte interactions are disrupted on ECs lacking CD44
We have demonstrated that HA and the standard form of CD44 on the luminal surface of CNS ECs contribute significantly to the initial capture and subsequent rolling of encephalitogenic lymphocytes in an inflammatory demyelinating disease model

Summary

Background

Multiple sclerosis (MS) is a demyelinating disease involving lymphocyte infiltration into the central nervous system (CNS). This process is initiated by L-selectin on lymphocytes and P- and E-selectins on ECs binding to their transmembrane glycoprotein ligands [4] These transient interactions result in lymphocyte rolling along the endothelial cell surface, enabling signaling that induces expression and activation of integrins that mediate firm adhesion [5]. There is evidence that P- and E-selectins are expressed by superficial blood vessels of the brain and that they can mediate rolling (6 – 8), evidence from both antibody blocking experiments and experiments with knock-out and transgenic mouse models suggest selectins and their ligands are not essential for the development of EAE [9, 10] These findings indicate the involvement of other adhesive molecules in lymphocyte rolling on vessels within the CNS. In vivo PEG-PH20 treatment delays the onset of EAE and reduces the number of T-cell infiltrates early in disease These data indicate that HA tethered to CD44 on ECs promotes lymphocyte rolling during EAE pathogenesis

EXPERIMENTAL PROCEDURES

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DISCUSSION