MATERIALS AND METHODS

Specific-pathogen-free BALB/cAnNCr female mice, 10-12 weeks old, were supplied by the Animal Production Area of the NCI-Frederick Cancer Research Facility. The mice were fed NIH-31 open formula diet and chlorinated water (15 ppm) ad libitum. A single 3-h exposure (8.6 X 10'J/m2) of UVB radiation was administered using a bank of 6 FS40 sunlamps (Westinghouse, Bloomfield, New Jersey), which delivered an average dose rate of 8.0 J/m2/s to the shaved dorsal surface of the mice [10]. During irradiation, the m1ce were confined in Plexiglas dividers, and the ears of those to be tested for CHS were covered with black electrical tape to protect the ears from radiation. AU sham-treated mice were shaved and treated identically except that they were not exposed to the UVB radiation. Blood from sham-treated or UVB-irradiated mice was obtained by retroorbital exsanguination after induction of anesthesia with metafane. The blood was allowed to clot at room temperature for 30 min, then centrifuged at 1000 g for 20 min at room temperature. The serum fraction from appropriate groups was pooled and transferred immediately as undiluted serum to normal recipient mice via tail vein injections.

On day 5 following treatment or serum transfer, the abdomens of all mice to be tested were razor shaved. Half of the mice in each group were then sensitized by painting the shaved area with 100 pl of 3% TNCB in acetone. The other half were painted only with vehicle (acetone). Seven days later, a challenge dose of 5 pi of 1% TNCB in acetone was applied to both surfaces of each ear on all mice. Ear thickness was measured prior to challenge and 24 h later with an engineer's micrometer (Swiss Precision Instruments, Inc., Los Angeles, California). This protocol has been shown to yield maximum response to TNCB in this laboratory (4]. CHS to TNCB was determined as the amount of ear swelling of sensitized mice minus the amount of ear swelling of nonsensitized mice from the same group and was designated 6. ear swelling. Each group consisted of 5 nonsensitized and 5 sensitized mice. All experiments were repeated at least 4 times and the data presented are consistent with each result. Spleens from appropriate groups were aseptically removed, teased in ice-cold Hanks' balanced salt solution (HBSS), pH 7.4, pooled, and passed through nylon mesh to yield single-cell suspensions. After 4 washes in cold HBSS, 1 X 108 viable nucleated spleen cells were transferred into normal mice via tail vein injection. Recipients were immediately sensitized to TNCB and challenged 7 days later, as described above.