FcγRIII-independent anaphylaxis in GαMD-primed mice requires challenge with a high dose of Ag. (A) BALB/c mice (5 per group) were primed s.c. with GαMD, then challenged i.v. 14 days later with 0.1 or 10 mg of GIgG. Some mice were pretreated 24 hours before GIgG challenge with 500 μg of anti–FcγRII/RIII mAb to block IgG-mediated anaphylaxis. Rectal temperatures were followed for 2 hours after challenge. (B) Mice primed and challenged as in A had blood drawn before and 15 minutes after challenge. Hematocrit levels were determined. *P < 0.05 compared with mice treated with anti–FcγRII/RIII mAb and challenged with 0.1 mg of GIgG. (C) WT (left) and FcγRIII-deficient mice (right) were primed s.c. with GαMD, then challenged i.v. 14 days later with 10 mg of GIgG. Some mice were injected s.c. with 500 μg of anti–FcγRII/RIII mAb 24 hours before GIgG challenge. Rectal temperatures were followed for 90 minutes after challenge. (D) BALB/c mice were primed s.c. with TNP-GαMD or saline, then challenged 14 days later with 0, 0.01, or 1 mg of biotinylated TNP-OVA. Blood was drawn 5 minutes later, and IgG1–TNP-OVA complexes in serum were quantitated by ELISA. *P < 0.05 compared with other measured levels. (E) TNP-OVA-NIP was diluted in nonimmune serum or heat-inactivated serum pooled from mice immunized 10–12 days earlier with GαMD (αGIgG Asm) or TNP-GαMD (αTNP Asm). Binding of serum TNP-OVA-NIP by IgEαTNP was measured by ELISA. Means ± SEMs are shown for all data in this and subsequent figures unless otherwise indicated.