Hyaluronic acid-CD44 interaction mediates the adhesion of lymphocytes by amniotic membrane stroma

Purpose: To demonstrate the role of intrinsic hyaluronic acid (HA) in the entrapment of inflammatory cells by amniotic membrane (AM) in vitro. Methods: HA concentration in AM was analyzed by the sandwich protein binding assay, and the approximate molecular weight was measured by gel filtration chromatography. To localize HA in AM tissue, toluidine blue staining with and without hyaluronidase treatment was performed. Adhesion of the lymphocyte cell lines, Jurkat and Molt 4, and peripheral blood mononuclear cells (PBMC) to AM and HA-coated glass slides was analyzed in an in vitro binding assay. Flow cytometry was performed to quantify the expression of the HA receptor, CD44, in Jurkat, Molt 4, and PBMC. Results: HA was present in high levels in the stroma of AM, also demonstrated by intense staining with toluidine blue. Staining was inhibited by both hyaluronidase treatment and acidic pH. Molt 4, which constitutively expressed CD44, bound to AM and HA-coated slides significantly more than Jurkat cells (CD44^-). Adhesion of Molt 4 was inhibited by pretreatment with both soluble HA and anti-CD44 antibody. LPS- or TNF-α-treated PBMC also bound to AM and HA-coated slides and was inhibited by pretreatment with an anti-CD44 antibody. Conclusion: HA in AM stroma may play an important role in the entrapment of inflammatory cells including lymphocytes when used as a patch in ocular surface disease.