Establishment of experimental eosinophilic vasculitis by IgE-mediated cutaneous reverse passive arthus reaction

Abstract

Prominent eosinophil infiltration is a characteristic of some forms of vasculitis, such as Churg-Strauss syndrome, also known as allergic granulomatous vasculitis. In the current study, we established a mouse model of cutaneous eosinophilic vasculitis by the cutaneous reverse passive Arthus reaction using IgE injection instead of IgG. Wild-type C57BL/6 mice were injected with IgE anti-trinitrophenyl antibodies, followed immediately by intravenous administration of trinitrophenyl bovine serum albumin. IgE-mediated immune complex challenge induced substantial hemorrhage with marked infiltration of eosinophils in which neutrophils, mast cells, and macrophages were also mixed. This finding contrasted remarkably with the neutrophil-dominant infiltration pattern in IgG-mediated immune complex challenge. In the lesion, the expression level of monocyte chemotactic protein-3 was increased, and anti-monocyte chemotactic protein-3 treatment resulted in a significant but incomplete blockade of eosinophil recruitment. Furthermore, mice lacking E-selectin, P-selectin, L-selectin, or intercellular adhesion molecule-1, as well as wild-type mice that received anti-vascular cell adhesion molecule-1-blocking antibodies were assessed for the IgE-mediated Arthus reaction. After 24 hours, the loss of P-selectin resulted in a significant reduction in eosinophil accumulation compared with both wild-type mice and other mouse mutants. Collectively, the Fc class of immunoglobulins, which forms these immune complexes, critically determines the disease manifestation of vasculitis. The IgE-mediated cutaneous reverse passive Arthus reaction may serve as an experimental model for cutaneous eosinophilic infiltration in vasculitis as well as in other diseases.

Figure 1

A: The macroscopic findings of IgE-mediated cutaneous reverse passive Arthus reaction in wild-type mice. B: The size of hemorrhage after 24 hours induced by intradermal IgG or IgE anti-TNP Ab injection followed by i.v. injection of TNP-BSA was assessed. The diameter ratio indicates the mean diameters of purpuric spots divided by the diameter of bleeding vessels. Horizontal bars indicate mean values for each group of mice.

Figure 2

Histological tissue sections of the skin from wild-type mice during IgE-mediated Arthus reaction. Skin tissues were harvested at 24 hours after IC challenge. H&E staining demonstrated a damaged vessel with prominent inflammation (A) and leukocytoclastic vasculitis with clearly visible eosinophils (B). Eosinophils (arrows) were detected by Hansel staining (C); neutrophils (arrows) by anti-MPO Ab staining (D); mast cells (arrows) by metachromatic staining of granules in toluidine blue-stained sections (E); and macrophages (arrows) by anti-F4/80 Ab staining (F). Original magnification = ×100 (A) and ×200 (B–F).

Figure 3

A, B: Time course of eosinophil (A) and mast cell (B) recruitment induced by IgE-mediated Arthus reaction in the skin tissue from wild-type mice. Mice were injected intradermally with IgE anti-TNP Ab, followed by i.v. TNP-BSA injection. The numbers of eosinophils and mast cells per skin section were determined by counting in Hansel- and toluidine blue-stained skin sections, respectively, before and at 4, 8, 12, 24, 48, and 72 hours after IC challenge. All values represent the mean ± SEM of results obtained from 5 to 10 mice. C, D, and E: Comparison of accumulated eosinophils (C), mast cells (D), and neutrophils (E) in the skin from wild-type mice between IgE- and IgG-mediated Arthus reaction at 24 hours after IC challenge. The numbers of eosinophils, mast cells, and neutrophils per skin section were determined by counting in Hansel-, toluidine blue-, and anti-MPO Ab-stained skin sections, respectively. All values represent the mean ± SEM of results obtained from 5 to 10 mice in each group.

Figure 4

Histological tissue sections showing eosinophil, neutrophil and mast cell accumulation of wild-type mice at 24 hours after IC challenge by intradermal injection of IgE (A) or (B) IgG anti-TNP. Hansel-staining (anti-Siglec-F Ab; insets) shows eosinophils (arrows; upper panels), and anti-MPO Ab staining (anti-Gr-1 Ab: insets) demonstrates neutrophils (arrows; middle panels). Mast cells were revealed by toluidine blue-staining (arrows; bottom panels). Original magnification = ×100 (toluidine blue) and ×200 (Hansel, anti-Siglec-F, anti-MPO, and anti-Gr-1).

Figure 5

mRNA expression levels of (A) eotaxin-1 and (B) MCP-3 in the dermis from wild-type mice before and at 12 and 24 hours after IgE anti-TNP Ab-mediated IC challenge. mRNA expression levels were assessed by real-time reverse transcription-PCR. Relative expression of real-time PCR products was determined by using the Ct method to compare target gene and house-keeping gene (GAPDH) mRNA expression levels. One of the control samples was chosen as a calibrator sample. The accumulation of eosinophils (C) and mast cells (D) was assessed in the skin from wild-type mice treated with polyclonal anti-MCP-3 Abs at 24 hours after IC challenge by intradermal injection of IgE anti-TNP Abs. All values represent the mean ± SEM of results obtained from 5 to 10 mice in each group.

Figure 6

Recruitment of (A) eosinophils and (B) mast cells in the skin from E-selectin (E-sel^−/−), P-selectin^−/− (P-sel^−/−), L-selectin^−/− (L-sel^−/−), and ICAM-1^−/− (ICAM^−/−) mice, wild-type mice treated with anti-VCAM-1 mAbs, and wild-type mice at 24 hours after IC challenge by intradermal injection of IgE anti-TNP Abs. The numbers of eosinophils and mast cells per section were determined by counting in Hansel- and toluidine blue-stained skin sections, respectively. C: Hemorrhages in these mice were evaluated by diameter ratio as shown in Figure 1. All values represent the mean ± SEM of results obtained from 5 to 10 mice in each group.