Figure 2.
Figure 2. IRBP- and S-Ag–induced leukocyte migration is specific and G-protein–coupled receptor mediated. (A) Antibodies against either bovine IRBP or S-Ag block lymphocyte migration. 10 (▧) or 100 (▪) ng/mL of IRBP were mixed with 20 μg/mL of rat polyclonal anti-IRBP prior to being placed in the lower wells of a micro-Boyden chemotaxis chamber. S-Ag (10, ▨, or 100, ▦, ng/mL) was mixed with 20 μg/mL of rabbit polyclonal anti–S-Ag prior to being placed in the lower wells of a micro-Boyden chemotaxis chamber. Lymphocytes cultured for 16 hours with IL-2 were placed in the upper wells. The mean number of migrating cells and corresponding standard deviation are graphed on the y-axis while chemoattractant and antibody conditions are shown on the x-axis. □ indicates media control. n = 3. A representative experiment is shown. (B) Both human and bovine S-Ag induce lymphocyte migration. Various concentrations of S-Ag were placed in the lower well of a micro-Boyden chemotaxis chamber. Human lymphocytes cultured for 16 hours with IL-2 were placed in the upper wells. The mean number of migrating cells and corresponding standard deviation are graphed on the y-axis while ligand concentrations are shown on the x-axis. ▧ represents human S-Ag; ▪, bovine S-Ag. n = 2. A representative experiment is shown. (C) Both S-Ag–and IRBP-induced lymphocyte migration is pertussis toxin sensitive. Human lymphocytes cultured for 16 hours with IL-2 were pretreated for 30 minutes at 37°C with increasing amounts of pertussis toxin. Various concentrations of chemoattractants were placed in the lower wells of a micro-Boyden chamber. Pertussis toxin–pretreated lymphocytes were placed in the upper wells. ▪ indicates S-Ag; ▨, IRBP; and ▦, regulated on activation, normal T-cell–expressed and secreted (RANTES). *StatView Student t test with P ≤ .0001 for maximum chemotactic responses vs “0” control; **P ≤ .005. Inhibited chemotaxis that was not significantly different from media control was noted with #. n = 3. Representative experiment is shown. Error bars indicate standard deviation.

IRBP- and S-Ag–induced leukocyte migration is specific and G-protein–coupled receptor mediated. (A) Antibodies against either bovine IRBP or S-Ag block lymphocyte migration. 10 (▧) or 100 (▪) ng/mL of IRBP were mixed with 20 μg/mL of rat polyclonal anti-IRBP prior to being placed in the lower wells of a micro-Boyden chemotaxis chamber. S-Ag (10, ▨, or 100, ▦, ng/mL) was mixed with 20 μg/mL of rabbit polyclonal anti–S-Ag prior to being placed in the lower wells of a micro-Boyden chemotaxis chamber. Lymphocytes cultured for 16 hours with IL-2 were placed in the upper wells. The mean number of migrating cells and corresponding standard deviation are graphed on the y-axis while chemoattractant and antibody conditions are shown on the x-axis. □ indicates media control. n = 3. A representative experiment is shown. (B) Both human and bovine S-Ag induce lymphocyte migration. Various concentrations of S-Ag were placed in the lower well of a micro-Boyden chemotaxis chamber. Human lymphocytes cultured for 16 hours with IL-2 were placed in the upper wells. The mean number of migrating cells and corresponding standard deviation are graphed on the y-axis while ligand concentrations are shown on the x-axis. ▧ represents human S-Ag; ▪, bovine S-Ag. n = 2. A representative experiment is shown. (C) Both S-Ag–and IRBP-induced lymphocyte migration is pertussis toxin sensitive. Human lymphocytes cultured for 16 hours with IL-2 were pretreated for 30 minutes at 37°C with increasing amounts of pertussis toxin. Various concentrations of chemoattractants were placed in the lower wells of a micro-Boyden chamber. Pertussis toxin–pretreated lymphocytes were placed in the upper wells. ▪ indicates S-Ag; ▨, IRBP; and ▦, regulated on activation, normal T-cell–expressed and secreted (RANTES). *StatView Student t test with P ≤ .0001 for maximum chemotactic responses vs “0” control; **P ≤ .005. Inhibited chemotaxis that was not significantly different from media control was noted with #. n = 3. Representative experiment is shown. Error bars indicate standard deviation.

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