Eosinophil pathogenicity mechanisms and therapeutics in neuromyelitis optica

Abstract

Eosinophils are abundant in inflammatory demyelinating lesions in neuromyelitis optica (NMO). We used cell culture, ex vivo spinal cord slices, and in vivo mouse models of NMO to investigate the role of eosinophils in NMO pathogenesis and the therapeutic potential of eosinophil inhibitors. Eosinophils cultured from mouse bone marrow produced antibody-dependent cell-mediated cytotoxicity (ADCC) in cell cultures expressing aquaporin-4 in the presence of NMO autoantibody (NMO-IgG). In the presence of complement, eosinophils greatly increased cell killing by a complement-dependent cell-mediated cytotoxicity (CDCC) mechanism. NMO pathology was produced in NMO-IgG-treated spinal cord slice cultures by inclusion of eosinophils or their granule toxins. The second-generation antihistamines cetirizine and ketotifen, which have eosinophil-stabilizing actions, greatly reduced NMO-IgG/eosinophil-dependent cytotoxicity and NMO pathology. In live mice, demyelinating NMO lesions produced by continuous intracerebral injection of NMO-IgG and complement showed marked eosinophil infiltration. Lesion severity was increased in transgenic hypereosinophilic mice. Lesion severity was reduced in mice made hypoeosinophilic by anti-IL-5 antibody or by gene deletion, and in normal mice receiving cetirizine orally. Our results implicate the involvement of eosinophils in NMO pathogenesis by ADCC and CDCC mechanisms and suggest the therapeutic utility of approved eosinophil-stabilizing drugs.

Figure 1. Eosinophils produce NMO-IgG–dependent ADCC and CDCC in cell cultures.

(A) Culture and characterization of eosinophils from murine bone marrow. Culture method shown at the left and MBPe immunofluorescence shown in the micrograph. Scale bar: 20 μm. (B) ADCC. AQP4-expressing CHO cells were incubated for 3 hours with 20 μg/ml NMO-IgG plus 300,000 eosinophils. Live/dead (green/red) staining is shown with percentage and density of live cells given below the micrographs (SEM, n = 10). Controls included untreated cultures, NMO-IgG or eosinophils alone, AQmab^ADCC plus eosinophils, and NMO-IgG plus eosinophils in nontransfected (AQP4-null) cells. Scale bar: 100 μm. (C) CDCC. Cells were incubated for 60 minutes with submaximal (5 μg/ml) NMO-IgG, 1% hc, and 100,000 eosinophils, with controls indicated. Percentage of dead cells quantified as in B (SEM, n = 10). Scale bar: 100 μm. Experiments were replicated 3 times. Eos, eosinophils; MPPs, multipotent progenitors.

Figure 2. Eosinophils produce NMO-IgG–dependent pathology by an ADCC mechanism in ex vivo spinal cord slice cultures.

(A) Vibratome-cut spinal cord slices from mouse were cultured on a porous support for 7 days, followed by the addition of NMO-IgG and/or cells for 1 day, and then stained for AQP4, GFAP, MBP, and Iba1. Immunofluorescence of spinal cord slices (from Aqp4^+/+ and Aqp4^–/– mice) were incubated as indicated (20 μg/ml NMO-IgG or AQmab^ADCC, 10⁷ eosinophils). Pathology scores are summarized at the bottom (SEM; 6–11 slices per condition; *P < 0.001 versus the control group; 1-way ANOVA). Scale bar: 500 μm. (B) Eosinophil degranulation by PAF produces NMO-like pathology (10⁷ eosinophils, 10 μg/ml PAF) (SEM; 5 slices per condition; *P < 0.01). Scale bar: 500 μm. Experiments were replicated 3 times.

Figure 3. Eosinophils produce NMO-IgG–dependent pathology by a CDCC mechanism in ex vivo spinal cord slice cultures.

(A) Spinal cord slice cultures prepared as in Figure 2 were incubated with submaximal NMO-IgG and hc as indicated (5 μg/ml NMO-IgG or AQmab^CDC, 3 × 10⁶ eosinophils, 5% hc). The experiments were conducted in parallel with those in Figure 2A using the same control slice group (SEM; 8–11 slices per condition; *P < 0.001 versus the control group; 1-way ANOVA). Scale bar: 500 μm. (B) Eosinophil degranulation by recombinant human C5a produces NMO-like pathology (10⁷ eosinophils pretreated with 5 μg/ml cytochalasin B, 30 ng/ml C5a) (SEM; 5 slices per condition; *P < 0.01). Scale bar: 500 μm. (C) Eosinophil toxin ECP produces NMO-like pathology (5 μg/ml NMO-IgG, 5% hc, 300 ng/ml ECP) (SEM; 4 slices per condition; *P < 0.01). Scale bar: 500 μm. Experiments were replicated 3 times.

Figure 4. In vivo mouse model of NMO with prominent eosinophil infiltration in lesions.

(A) Mice were administered NMO-IgG and hc by 3-day continuous intracerebral injection using an implanted minipump. (B) Immunofluorescence staining of brain sections after 3-day infusion with NMO-IgG (3.3 μg/day) and complement (16.7 μl/day) stained for astrocyte markers AQP4, GFAP, and EAAT1, myelin marker MBP, and leukocyte marker CD45, in Aqp4^+/+ and Aqp4^–/– mice. White dotted lines demarcate the area of staining loss. Scale bar: 2 mm. (C) Higher magnification showing AQP4 and EAAT1, MBP, activated complement marker C9neo, markers of eosinophils (Siglec F) and neutrophils (Ly6G), and H&E staining in the lesion core, periphery, and contralateral side. Scale bars: 100 μm. Arrowhead indicates eosinophils in high magnificent H&E staining. Scale bar: 20 μm. (D) Percentage of area of loss of AQP4 staining (SEM; 3–6 mice per group; *P < 0.05). Mice were infused for 3 days as in B with NMO-IgG alone, control IgG plus complement, or IgG (66 μg/day) purified from 3 different NMO sera. (E) Immunofluorescence of AQP4, C9neo, Siglec F, and Ly6G after 1 day of minipump infusion (representative of 3 mice). Scale bar: 100 μm.

Figure 5. Eosinophil-dependent NMO pathology in mice.

(A) Mice were intraperitoneally administered anti–IL-5 or anti-Ly6G antibodies, alone or together, to deplete eosinophils and/or neutrophils, followed by a 3-day NMO-IgG/hc infusion (top). Peripheral neutrophil (Nϕ) and eosinophil counts in control and treated mice (bottom) (SEM; 5 mice per group; P < 0.001). (B) Mice were infused for 3 days with 3.3 μg/day NMO-IgG and 16.7 μl/day hc, and sacrificed on day 3. Immunofluorescence staining of AQP4 and MBP (scale bar: 2 mm); Siglec F and Ly6G (scale bar: 100 μm). (C) Lesion severity assessed by loss of AQP4, myelin, and numbers of eosinophils and neutrophils in lesions (SEM; 4–8 mice per group; *P < 0.05; **P < 0.01). (D) Control and hypereosinophilic IL-5 Tg mice were infused for 3 days with NMO-IgG (3.3 μg/day) and submaximal hc (3.4 μl/day). Immunofluorescence staining of AQP4 (scale bar: 2 mm), Siglec F, and Ly6G (scale bar: 100 μm). Summary of lesion scores (SEM; 4 mice per group; *P < 0.05). (E) Control and Tg hypoeosinophilic ΔdblGata1 mice were infused for 3 days with NMO-IgG (3.3 μg/day) and hc (16.7 μl/day). AQP4 immunofluorescence and loss of AQP4 immunofluorescence are shown (SEM; 5 mice per group; *P < 0.05). Scale bar: 2 mm.

Figure 6. Small-molecule eosinophil inhibitors reduce NMO-IgG–dependent cytotoxicity and NMO lesions.

(A) Reduced EPO release following 30-minute incubation of eosinophils with 5 μM PAF and indicated inhibitors (representative of 3 sets of experiments). Inset shows dose response for cetirizine (at 5 μM PAF, n = 6) (left). Summary of EPO release data (SEM; n = 6) (center). Lack of compound effect on NMO-IgG–mediated CDC in AQP4-expressing CHO cells (SEM; n = 8) (right). (B) EPO release into media for AQP4-expressing CHO cells treated for 1 hour with 10⁶ eosinophils without or with 20 μg/ml NMO-IgG. Where indicated, 5 μM cetirizine was present (SEM; n = 6; *P < 0.05). (C) Spinal cord slice cultures were exposed to NMO-IgG without complement (top, ADCC), or with complement (bottom, CDCC), and without or with eosinophils, as done in Figures 2 and 3. Where indicated, cetirizine (1 μM), ketotifen (50 μM), or IBMX (300 μM) were present during the 24-hour incubation. Scale bars: 500 μm. (D) Intracerebral infusion was done (as in Figure 5) without versus with cetirizine administration (10 mg/kg b.i.d., 1 day prior to and during 3-day infusion). Immunofluorescence staining is shown on the top and lesion scores are summarized on the bottom (SEM; n = 4–6; *P < 0.05). Scale bars: 2 mm; 50 μm. (E) Chemical structures of cetirizine and analogs (left). Summary of EPO release data (SEM; n = 6) (center). Summary of data from intracerebral infusion model (right) (each compound was given 10 mg/kg b.i.d.) (SEM; n = 3–7 for each group; *P < 0.05).

Figure 7. Proposed mechanism of eosinophil-dependent NMO pathogenesis.

Eosinophils produce complement-independent and -dependent astrocyte damage. ADCC (left) involves binding of NMO-IgG to AQP4 on astrocytes, which causes eosinophil binding (involving Fcγ receptors) and degranulation. The released granule toxins damage astrocytes. Astrocyte damage is amplified when complement is present by CDCC (right), which involves multiple mechanisms, including complement-dependent enhancement of eosinophil binding to AQP4-IgG and increased eosinophil accumulation and degranulation.