A NEW METHOD FOR STUDYING THE SELECTIVE ADHERENCE OF BLOOD-LYMPHOCYTES TO THE MICROVASCULATURE OF HUMAN RETINA

Purpose. To develop a sensitive and reproducible technique for measuri ng the adherence of blood lymphocytes to vessel walls exposed in secti ons of human retina and for examining the role of lymphocyte and vascu lar adhesion molecules in these events. Methods. Cryostat sections of human retina were overlaid wi th blood lymphocytes from healthy subjec ts, and experimental conditions were sought by which preferential atta chment of the cells occurred to blood vessel walls in the retinal sect ions. Adherent lymphocytes were identified by staining with methyl gre en-thionine, and transected blood vessels were identified by their str ucture and by staining of basement membranes with periodic acid-Schiff . The adherence of enriched preparations of CD4(+) (T-helper) and CD8( +) (T-cytotoxic) lymphocytes, of interleukin-2 (IL-2)-activated cells, and of lymphocytes from patients with ocular Behcet's disease was exa mined. The distribution of adhesion molecules on retinal vessel walls was determined by immunohistochemistry, and the contribution of leukoc yte integrins to lymphocyte binding was studied by blocking experiment s with monoclonal antibodies. Results. The optimal selectivity of bloo d lymphocyte attachment to retinal vessel walls occurred when purified lymphocytes were suspended in culture medium with 10% fetal calf seru m and overlaid onto retinal sections for 30 minutes at 23 degrees C wi th gentle agitation. Under these conditions, 92% of the lymphocytes th at adhered to the section were confined to the retinal microvasculatur e, and CD4(+) T cells were more adherent than CD8(+) T cells (P < 0.01 ). Prior exposure of normal lymphocytes to IL-2 enhanced their binding to retinal blood vessels, and lymphocytes from patients with Behcet's disease showed supranormal vascular adherence (P < 0.005). Many trans ected vessels stained positively for CD31; PECAM (mean 62%), CD54; ICA M-1 (mean 73%), CD62E; E-selectin (mean 35%), CD62P; P-selectin (mean 61%), and CD106; VCAM-1 (mean 42%). However, these vascular adhesion m olecules occupied <20% of the area of the blood vessel walls. Lymphocy te adhesion to the retinal vessels was more dependent on CD29 (the com mon chain of the pr integrins) expression than either CD11a/CD18 or CD 49d. Conclusions. This technique allows measurements to be made of lym phocyte adherence to vascular and nonvascular structures of retina ex vivo. Extension of this approach to the study of leukocyte adherence t o sections of pathologic retina may be of clinical and experimental ap plicability in understanding mechanisms of retinal inflammation.