EAU was induced by active immunization with graded doses of IRBP
or p161-180 in phosphate-buffered saline (PBS) emulsified 1:1 vol/vol
in complete Freund’s adjuvant (CFA) that had been supplemented with
Mycobacterium tuberculosis strain H37RA (Sigma, St. Louis,
MO) to 2.5 mg/ml. A total of 200 μl emulsion was injected
subcutaneously, divided among three sites: base of tail and both
thighs. In some groups, 0.5 μg of
Bordetella pertussis toxin (PTX) (Sigma) was injected intraperitoneally at the same time.
EAU by adoptive transfer was induced by intraperitoneal injection of
pooled spleen and lymph node cells obtained from primed donors and
stimulated in culture with 20 μM p161-180 in the presence or absence
of 50 ng/ml interleukin (IL)-12, essentially as previously
described.
15 In some adoptive transfer recipients, 1 μg
PTX was administered intravenously just before the adoptive transfer.
Clinical EAU was evaluated by fundoscopy under a binocular microscope
after dilation of the pupil and graded on a scale of 0 to 4 using
criteria described in detail elsewhere.
16 Eyes harvested
21 days after immunization or 10 days after adoptive transfer were
prefixed in 4% phosphate-buffered glutaraldehyde for 1 hour (to
prevent artifactual detachment of the retina), and then transferred to
10% phosphate-buffered formaldehyde until processing. Fixed and
dehydrated tissue was embedded in methacrylate, and 4- to 6-μm
sections were stained with standard hematoxylin and eosin. Eye sections
cut at different planes were scored in a masked fashion. Incidence and
severity of EAU were graded on a scale of 0 to 4 in half-point
increments, using the criteria described previously,
16 which are based on the type, number, and size of lesions present.
Incidence was shown as the number of positive animals of all animals in
the group. Severity of disease was the average score of eyes from those
animals in which disease developed (if disease was unilateral, both
eyes were averaged).