IgG-blocking antibodies inhibit IgE-mediated anaphylaxis in vivo through both antigen interception and Fc gamma RIIb cross-linking

Abstract

Although it has long been hypothesized that allergen immunotherapy inhibits allergy, in part, by inducing production of IgG Abs that intercept allergens before they can cross-link mast cell Fc epsilonRI-associated IgE, this blocking Ab hypothesis has never been tested in vivo. In addition, evidence that IgG-allergen interactions can induce anaphylaxis by activating macrophages through Fc gammaRIII suggested that IgG Ab might not be able to inhibit IgE-mediated anaphylaxis without inducing anaphylaxis through this alternative pathway. We have studied active and passive immunization models in mice to approach these issues and to determine whether any inhibition of anaphylaxis observed was a direct effect of allergen neutralization by IgG Ab or an indirect effect of cross-linking of Fc epsilonRI to the inhibitory IgG receptor Fc gammaRIIb. We demonstrate that IgG Ab produced during the course of an immune response or administered passively can completely suppress IgE-mediated anaphylaxis; that these IgG blocking Abs inhibit IgE-mediated anaphylaxis without inducing Fc gammaRIII-mediated anaphylaxis only when IgG Ab concentration is high and challenge allergen dose is low; that allergen epitope density correlates inversely with the allergen dose required to induce both IgE- and Fc gammaRIII-mediated anaphylaxis; and that both allergen interception and Fc gammaRIIb-dependent inhibition contribute to in vivo blocking Ab activity.

Figure 1

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.

Figure 2

IgE/FcεRI/mast cell–dependent anaphylaxis in GαMD-primed mice requires challenge with a high dose of Ag. Mice (4–5 per group) were primed s.c. with 0.2 ml of GαMD, then challenged i.v. 14 days later with GIgG. Temperature was followed for 2 hours after challenge, and the maximum temperature decrease was calculated. Mice were matched for genetic background in all experiments. (A) WT mice and mice deficient in FcγRIII, IgE, or both were challenged as shown. (B) WT (+) and mast cell–deficient W/W^v (–) mice were treated as shown. (C) BALB/c mice were injected 15–30 minutes before challenge with 66 μg of CV6209 (PAF antagonist), 0.2 mg of both triprolidine and cimetidine (H1 and H2 antagonists), all 3 antagonists, or no antagonist and challenged as shown. (D) BALB/c mice were injected i.v. with 1 mg of gadolinium (macrophage inhibitor) or saline 1 day before GIgG challenge. (E) BALB/c mice were injected s.c. with saline or 500 μg of anti–FcγRII/RIII mAb 1 day before GIgG challenge. Blood was drawn 2 hours after GIgG challenge, and MMCP-1 levels were determined. (F) BALB/c mice were injected s.c. with saline or 500 μg of anti–FcγRII/RIII mAb 1 day before GIgG challenge. Anticoagulated blood was obtained for histamine measurement 5 minutes after challenge. (G) BALB/c mice were bled 4 hours after challenge with the indicated dose of GIgG, and IL-4 secretion was evaluated by in vivo cytokine capture assay (IVCCA) (51). *P < 0.05.

Figure 3

Identification of the serum factor that blocks IgE-mediated anaphylaxis as Ag-specific IgG. (A) BALB/c mice (5 per group) were primed with 10 μg of IgEαTNP i.v., then challenged i.v. 24 hours later with the doses of TNP-OVA shown on the abscissa. Maximum temperature decreases during the 90 minutes after challenge were calculated for this and all subsequent panels. (B) BALB/c mice (5 per group) were primed i.v. with the doses of αTNP Asm shown on the abscissa and challenged i.v. 24 hours later with the indicated doses of TNP-OVA. (C) BALB/c mice (5 per group) were primed i.v. with 10 μg of IgEαTNP, 250 μl of αGIgG Asm, and/or 250 μl of αTNP Asm as indicated, and challenged i.v. 24 hours later with 1 μg of TNP-OVA. *P < 0.05. (D) BALB/c mice (5 per group) were primed i.v. with 10 μg of IgEαTNP plus saline, 250 μl of IgGαTNP, or 125 μl of αTNP Asm, then challenged i.v. 24 hours later with 70 ng of TNP-OVA. (E) BALB/c mice (5 per group) were primed i.v. with 250 μl of IgGαTNP, then challenged i.v. 24 hours later with 70 ng or 500 μg of TNP-OVA. (F) BALB/c mice (5 per group) were primed i.v. with either 10 μg of IgEαTNP or 250 μl of αTNP Asm or both and treated with saline or 500 μg of anti–FcγRII/RIII mAb. Mice were challenged i.v. 24 hours later with 1 or 500 μg of TNP-OVA.

Figure 4

Effects of Ag epitope density on IgE- and FcγRIII-mediated anaphylaxis and IgG BA inhibition of IgE-mediated anaphylaxis. (A) BALB/c mice (5 per group) were primed i.v. with either 10 μg of IgEαTNP (left) or 40 μl of αTNP Asm (right), then challenged i.v. 24 hours later with TNP-OVA. Doses of TNP-OVA conjugates are indicated on graph abscissas; molar TNP/OVA ratios of the different conjugates tested are indicated in the figure. Maximum temperature decreases during the 90 minutes after challenge were determined. (B) BALB/c mice (5 per group) were primed i.v. with 10 μg of IgEαTNP and injected i.v. with the quantities of αTNP Asm indicated on the graph abscissas. Mice were injected i.v. 24 hours later with 10 μg of biotin–anti–IL-4 mAb and challenged i.v. with the indicated doses of the TNP-OVA conjugates. Maximum temperature decreases during the 90 minutes after challenge were determined (left). Blood was drawn 2 hours after challenge, and IL-4 secretion was evaluated by IVCCA (right) (51).

Figure 5

IgG BA inhibits IgE-mediated anaphylaxis through both FcγRIIb-dependent and -independent mechanisms. (A) WT and FcγRIIb-deficient mice (8–10 per group) were primed i.v. with 10 μg of IgEαTNP and treated i.v. with the quantities of αTNP Asm indicated on the graph abscissas. Mice were injected i.v. 24 hours later with 10 μg of biotin–anti–IL-4 mAb and challenged i.v. with 100 ng of TNP-OVA. Maximum temperature decreases during the 90 minutes after challenge were determined. Blood was drawn 2 hours after challenge, and IL-4 secretion was determined by IVCCA. All mice survived. (B) FcγRIII-deficient mice (5 per group) were primed i.v. with 10 μg of IgEαTNP and treated i.v. with the quantities of αTNP Asm indicated on the graph abscissas and s.c. with 500 μg of either anti–FcγRII/RIII mAb or isotype-matched control mAb. Mice were injected i.v. 24 hours later with 10 μg of biotin–anti–IL-4 mAb and challenged i.v. with 1 μg or 100 ng of TNP-OVA. Maximum temperature decreases during the 90 minutes after challenge were determined. Survival was 100% for all mice challenged with 100 ng of TNP-OVA and as indicated for mice challenged with 1 μg of TNP-OVA. Blood was drawn 2 hours after challenge, and IL-4 secretions were determined by IVCCA for mice challenged with 100 ng TNP-OVA. *P < 0.05. ^†P < 0.05 compared with control mAb–treated mice that received no αTNP Asm.