Biphasic effects of cyclophosphamide on l-asparaginase hypersensitivity and resistance in mice Free

Background: L-Asparaginase (ASNase), a crucial drug for childhood acute lymphoblastic leukemia (ALL) treatment, frequently causes allergic reactions and drug resistance. Although the incidence rate of ASNase-induced hypersensitivity reactions seems to be higher than food allergies, their characteristics and exacerbating factors remain poorly understood. Furthermore, while certain anti-cancer drugs affecting lymphocyte functions are often co-administered with ASNase, their roles in ASNase hypersensitivity and drug resistance are unknown.

Aims: This study aimed to establish in vivo and in vitro models of ASNase-induced type I hypersensitivity in mice to investigate its mechanism and identify potential treatments. We evaluated an anti-IgE antibody, which targets the Fc region of IgE to block its binding to the FcεRI receptor, as a therapeutic candidate. Additionally, we examined the effects of methotrexate (MTX) and cyclophosphamide (CY), drugs known to impair Th17 and Treg cell functions, respectively, on ASNase allergy and drug resistance.

Methods: Male BALB/c mice were sensitized with ASNase and Al(OH)3 gel on days 0 and 14. The right ears of the mice were locally sensitized on days 17, 20, 23 by intradermally (i.d.) injection of ASNase. Antigen challenge was carried out on day 26 by i.d. injection. MTX (1, 10, 100 mg/kg) or CY (75, 150, 300 mg/kg) was intraperitoneally (i.p.) injected on days -2 and 12. An anti-IgE antibody (50, 100 μg/mouse) was i.p. injected on day 25. Serum was collected on day 26 to measure total IgE levels by ELISA and ASNase activity using an assay kit. RBL-2H3 cells were sensitized with collected serum and stimulated with ASNase to quantify β-hexosaminidase (β-Hex) release in vitro. Spleen cells from sensitized mice were cultured with concanavalin A for 48 hours, and Th1 and Th2 cytokines in the supernatant were measured using a Bio-Plex assay kit.

Results: ASNase administration induced ear edema and increased serum total IgE levels in mice. Anti-IgE antibody significantly reduced both ear swelling and serum total IgE levels. Cyclophosphamide (CY) at 150 mg/kg enhanced ASNase-induced allergic ear edema and increased serum IgE. Conversely, CY at 300 mg/kg increased serum total IgE but decreased ASNase-induced ear edema and serum ASNase activity. Methotrexate (MTX) had no effect on ASNase allergy or serum IgE levels. In vitro, ASNase challenge induced β-Hex release from RBL-2H3 cells sensitized with anti-ASNase serum. Serum from CY 150 mg/kg-treated mice induced higher β-Hex release, whereas serum from CY 300 mg/kg-treated mice did not. However, after IgG removal from the serum of CY 300 mg/kg-treated mice, β-Hex release increased to levels higher than normally sensitized mice. Cytokine measurements from spleen cell supernatants showed that ASNase sensitization increased IL-4, IL-12, and IL-10 production. CY further promoted IL-4 and IL-12 production in a dose-dependent manner. The IL-12/IL-4 ratio decreased in ASNase-sensitized mice, and CY co-administration further lowered this ratio, indicating strong Th2-biased immune promotion by CY. CY at 300 mg/kg also strongly enhanced IL-10 production.

Conclusions: We successfully established in vivo and in vitro models for Th2-biased type I hypersensitivity to ASNase. Our findings suggest that non-anaphylactogenic neutralizing IgE antibodies, such as omalizumab, are promising candidates for treating ASNase hypersensitivity. Furthermore, CY administration exhibited biphasic effects on ASNase allergy in sensitized mice: enhancing it at lower dosages (150 mg/kg) by promoting Th2-biased immune responses and increasing IgE production, but attenuating it at higher dosages (300 mg/kg). The high dose of CY also reduced ASNase activity and induced drug resistance. The elevated IL-10 levels observed with high-dose CY, alongside high IL-4, may contribute to the class switching from IgE to IgG, potentially explaining the attenuated allergic response despite increased IgE. MTX did not affect the immune reaction to ASNase. These results collectively emphasize that understanding the immunological impact of concomitant drugs is crucial for managing the risks of ASNase-induced allergy and drug resistance.