Hyaluronic acid-CD44 interaction mediates the adhesion of lymphocytes by amniotic membrane stroma.
OBJECTIVE
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-alpha-treated PBMC also bound to AM and HA-coated slides and was inhibited by pretreatment with an anti-CD44 antibody.


CONCLUSIONS
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.
Higa
Kazunari
K
Cornea Center, Ichikawa General Hospital, Tokyo Dental College, Chiba, Japan. higa@eyebank.or.jp
Shimmura
Shigeto
S
Shimazaki
Jun
J
Tsubota
Kazuo
K
D002448:Cell Adhesion / Q000187:drug effects / Q000502:physiology; D002460:Cell Line; D002850:Chromatography, Gel; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D018960:Hyaluronan Receptors / Q000378:metabolism*; D006820:Hyaluronic Acid / Q000378:metabolism*; D019169:Jurkat Cells / Q000378:metabolism; D008024:Ligands; D008070:Lipopolysaccharides / Q000494:pharmacology; D009000:Monocytes / Q000378:metabolism; D011720:Pyrazoles / Q000378:metabolism*; D013601:T-Lymphocytes / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D018960:Hyaluronan Receptors; D008024:Ligands; D008070:Lipopolysaccharides; D011720:Pyrazoles; D014409:Tumor Necrosis Factor-alpha; C012724:aminon; D006820:Hyaluronic Acid