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S. Rathod et al.
hypersensitivity reaction.2,7 Interestingly, few studies have assessed anti-ASNase IgE levels in patients or have sug- gested a role of anti-ASNase IgE in the ASNase hypersen- sitivity reaction,14-16 likely due to a lack of methods avail- able to detect anti-ASNase IgE in the presence of high anti- ASNase IgG levels.
Pharmacogenomic studies of the immune response to ASNase have identified risk variants in genes involved in antigen presentation17 or T-cell activation,18 supporting the importance to B/T-cell activation during ASNase sensitiza- tion and in the production of anti-ASNase antibodies. Anti-ASNase IgG can elicit a hypersensitivity reaction by forming an immune complex with ASNase, binding to the Fcγ receptor of immune cells (e.g., basophils, mast cells, neutrophils, and/or macrophages), and resulting in the release of platelet activating factor (PAF).19 Alternatively, cells expressing FcεRI, such as mast cells and basophils, can bind anti-ASNase IgE during sensitization and medi- ate a hypersensitivity upon antigen exposure via the release of histamine.20 Recently, a murine model of ASNase hypersensitivity has been described that recapitu- lates many of the features of clinical ASNase-mediated hypersensitivity.21 Studies using this model indicate that pretreatment with the antihistamine, triprolidine, and the PAF receptor antagonist, CV-6209, can strongly mitigate the onset of ASNase hypersensitivity, suggesting that both histamine and PAF release play a role in the immune response to ASNase.
The current study uses the murine model of ASNase hypersensitivity to identify the immune cells required for ASNase sensitization and the immunoglobulin isotypes and receptors responsible for the onset of hypersensitivity. Our results indicate that anti-ASNase IgE plays an impor- tant role in ASNase-induced hypersensitivities and that the binding of ASNase to basophils may be predictive of hypersensitivity. In accordance with the importance of binding to basophils, we show that ASNase activates basophils through both FcγRIII- and IgE-dependent mech- anisms. Our results suggest that both anti-ASNase IgG and IgE play important roles in the development of ASNase hypersensitivity.
Methods
ASNase sensitization
8-week-old, female, C57BL/6 mice were injected intraperitoneally (IP) with 10 mg of E. coli ASNase formu- lated with 1 mg of aluminum hydroxide adjuvant, on days 0 and 14, as previously described.21 ASNase hypersensitiv- ity reactions were induced in sensitized mice by challeng- ingwitha100mgIVdoseofE.coliASNaseonDay24of treatment. All experiments with mice were reviewed and conducted under approved protocol by the University of Pittsburgh Institutional Animal Cares and Use Committee.
Basophilic activation test (BAT)
BAT was performed as previously described.22,23 Briefly, 50 mL of blood was incubated for 15 min at 37°C and fur- ther stimulated with EM-95 at 300 ng/mL, 2.4G2 at 300 ng/mL, ASNase at 1 IU/mL, or medium (as a negative con- trol). Samples were further incubated for 2 h at 37°C in 5% CO2, quenched by adding 20 mM EDTA, and incu- bated on ice for 10 minutes. Cells were blocked with 15% HS in PBS for 30 minutes on ice, washed, and stained with anti-IgE, anti-CD49b, anti-CD200R3, and anti-CD200R1 mAbs for 30-60 minutes at 4°C. The cells were then lysed, washed with 1% BSA in PBS, and analyzed by flow cytometry. The percent change in CD200R1 expression is equal to the mean experimental expression of CD200R1 minus that of the mean expression of the sample stimulat- ed with medium, divided by the mean expression of the sample stimulated with medium. Similarly, the percent change in CD200R3 is the mean expression of the sample stimulated with medium minus the mean experimental expression of CD200R3, divided by the mean expression of the sample stimulated with medium.
Detection of anti-ASNase IgE by flow cytometry
In vivo blocking of ASNase-induced hypersensitivity reactions with anti-IgE or anti-FcγRIIB/III mAb
To prevent IgE- or IgG-mediated hypersensitivities, a single 100 μg dose of anti-IgE (EM-95)20 or 500 μg of anti- FcγRIIB/III mAb (2.4G2)24 was administered IP 24 hours before the ASNase challenge. Pretreatment medication, as a single agent or in combination, was given before the ASNase challenge in a total volume of 150 mL per injec- tion. The doses of each drug used are based on previous studies.21 66 mg of CV-6209 (PAF receptor antagonist) was given 5 minutes before challenges via IV injection, and 200 mg of antihistamine (triprolidine, an H1 receptor antago- nist) was given IP 30 minutes before the ASNase chal- lenge. Additional Methods are included in the Online SupplementaryMaterial.
Results
Anti-ASNase IgE plays a role in ASNase-specific recognition
The onset of ASNase-mediated hypersensitivity requires a humoral immune response to the agent after its initial use during induction therapy and antigen-specific recognition upon drug re-exposure after sensitization. Therefore, as expected, staining peripheral blood cells from naïve mice with fluorochrome-labeled ASNase (Figure 1A) revealed no ASNase positive neutrophils, T cells, basophils, or B cells. Yet, consistent with the per- ceived mechanism of ASNase clearance involving macrophages of the reticuloendothelial system (RES),25 a small percentage of macrophages/monocytes were ASNase positive (Figure 1A). Mice were given 100 mg of fluorochrome-labeled ASNase IV to verify its uptake by macrophages/monocytes. The labeled ASNase was prima-
Anti-IgE-biotin (Biolegend, USA) at 1 mg/mL was bound to 3x106 streptavidin-coupled 6-8 mm diameter magnetic particles (Spherotech, USA). Plasma samples diluted to 1:100 in PBS were added to anti-IgE-coated beads for 30- 60 minutes at room temperature, washed with PBST, and stained with labeled ASNase at 1 IU/mL. The stained sam- ples were analyzed by flow cytometry for ASNase fluo- rescence.
In vivo immune cell depletion
Anti-CD4 mAb or anti-CD19 mAb were injected IP in
mice at 200 mg/mouse three days before each sensitization dose of ASNase. Cell depletions were confirmed by flow cytometry, as described above, where different mAb clones targeting CD19 or CD4 were used for cell depletion and staining. Mice were challenged with E. coli ASNase on Day 24, as described above.
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haematologica | 2019; 104(2)