Hr44 Secreted with Exosomes: Loss from Ciliary Epithelium in Response to Inflammation

Hr44 is a target antigen for cross-reactive antibodies to the Onchocerca volvulus antigen Ov39. This study was undertaken to determine the epitopes recognized by antibodies to hr44, including Ov39/hr44 cross-reacting antibodies, and to use these antibodies to determine the distribution of hr44 in normal and inflamed intraocular epithelia and ARPE-19 cell cultures. Epitopes were identified with a peptide-based ELISA and competition ELISA. Immunostaining for hr44 and CD63 was conducted on control and inflamed ocular tissues. Exosomes were isolated from ARPE-19 cell cultures and analyzed by Western blot and electron microscopy. Linear epitopes and the Ov39 cross-reactive epitope of hr44 were identified. Immunohistology indicated that hr44 is present in vesicular structures of the iris and ciliary body and is lost from the epithelial layers of inflamed eyes coincidentally with CD63. A 66-kDa variant of hr44 is present in exosomes purified from ARPE-19 cell culture supernatants. Because hr44 is a component of exosomes produced by ARPE-19 cells, the coincident loss of hr44 and CD63 in inflamed epithelia indicates that exosomes may be released from intraocular epithelia in response to inflammation. This notion suggests that exosomes shed by intraocular epithelial cells influence T-cell survival and antigen presentation in the eye without direct cell-cell contact and have a role in the maintenance of ocular immune privilege.