1960. not recommended for humans because of concerns associated with whole-cell vaccines (22). However, Crolibulin the ability of whole cells to consistently induce arthritis in our murine model (8, 10) allowed for the evaluation of the immunological mechanisms that induce arthritis. Infection of mice. Twenty-one days after vaccination, mice were anesthetized with ether contained in a nose-and-mouth cup and injected subcutaneously in the right hind paw with 50 l of BSK medium containing 106 viable organisms. Swelling of the hind paws consistently develops 4 to 6 6 days after infection and peaks on day 8 to 12 (8, 10). Swelling of the hind paws can also be induced by infection with the homologous strain 297. However, strain 297-vaccinated mice must be challenged before protective antibodies develop (approximately day 7) or after their decline. Crolibulin Swelling of the hind paws of homologous vaccinated and challenged mice is variable. Therefore, we challenged strain 297-vaccinated mice with to obtain consistent swelling of the hind Crolibulin paws. Vaccination of mice with and challenge with strain 297 also yields consistent swelling of the hind paws, as does challenge with other infectious isolates of (11, 27, 37). Controls included vaccinated mice injected with alum or BSK medium alone. Administration of anti-IL-15 antibody and rIL-15 receptor alpha. Lyophilized goat anti-mouse immunoglobulin G polyclonal IL-15 antibody (200 g), normal goat immunoglobulin G (100 g), and mouse rIL-15 receptor alpha (100 g) were obtained from R&D Systems (Minneapolis, MN). The antibodies and rIL-15 receptor were resuspended in filter-sterilized (0.2-m-pore-size filter) (Acrodisk; Gilman Sciences, Ann Arbor, MI) PBS (pH 7.2) or PBS containing 0.1% bovine serum albumin (Fisher Scientific, Pittsburgh, PA), respectively, to yield concentrations of 50 g/ml. Twenty-one days after vaccination, three groups of eight mice each were infected with 106 organisms in the right hind paw. Less than 1 h after infection, the mice were injected subcutaneously in the right hind paw with 50 l of GCN5L the anti-IL-15 antibody or rIL-15 receptor alpha preparation. Anti-IL-15 antibody or rIL-15 receptor alpha was injected daily for 6 or 8 days, respectively. In other experiments, anti-IL-15 antibody was injected on day 7 after infection and daily thereafter for 6 days. Control groups received injections with the normal goat isotype antibody or with BSK medium. Measurement of IL-17 produced by immune lymph node cells. Twenty-one days after vaccination, six mice were euthanized with ether contained in a nose-and-mouth cup and the inguinal lymph nodes were removed. The nodes were teased apart with a forceps, and single-cell suspensions were obtained by passing the cells through a sterile Falcon 100-m nylon cell strainer (Fisher Scientific) into cold RPMI medium containing 10% fetal calf serum (Sigma, St. Louis, MO) with penicillin and streptomycin (Fisher Scientific). The cells were counted by using a hemacytometer and dispensed at a concentration of 5 106 cells per well into a 24-well microtiter plate (Fisher Scientific) in 1 ml of supplemented RPMI medium. Mouse rIL-15 ( 1.0 endotoxin unit of endotoxin per 1 g cytokine; R&D Systems) was reconstituted in PBS containing 0.1% bovine serum albumin and added to wells at a concentration of 50, 100, 200, or 500 ng/well. Wells not receiving rIL-15 were treated with PBS containing 0.1% bovine serum albumin. Viable organisms (5 106) were added to some wells. Microtiter plates were incubated at 37C in 5% CO2 for 48 h before supernatants were removed and analyzed for production of IL-17 using an enzyme-linked immunosorbent assay kit (R&D Systems) according to the manufacturer’s instructions. Assessment of arthritis. Hind-paw swelling was used to determine the level of the inflammatory response in the study mice. Prior to vaccination, random age-matched mice were chosen and their right hind paws were measured to determine the baseline of paw size. Paws were measured 48 h after infection and every other day thereafter for 14 days by using a Vernifer caliper with a digital readout to 0.01 mm. To obtain the measurements, mice were anesthetized with ether and the widths and thicknesses of the right hind tibiotarsal joints were carefully measured. All caliper values within a group were summed and divided by the number of measurements taken in the group to obtain the daily mean value. Preparation of tissues for histologic examination. At 9 and 14 days after infection, mice were euthanized with ether and their hind paws were.