The effects of treatment with IL-5 after the onset of clinical EAN. (A) rIL-5 treatment (5000 U/d for 10 days; ●; n = 15) commenced on the day after onset of clinical EAN, usually day 12, reduced severity of EAN within 2 days, compared with sham-treated (gray circle; n = 10) or untreated (○; n = 13) controls. In contrast to both sham and untreated controls, rIL-5–treated animals had significantly lower clinical scores between day 15 and day 21 (P < .008) and greater weight loss between day 14 and day 26 (P < .03). At days 14 and 21 after immunization, nerves were examined. (B) Electromicrographs (bar represents 1 μm) showed major disruption with numerous demyelinated nerves and edema with mononuclear cell infiltrates in controls, whereas rIL-5–treated animals had well-preserved myelin sheaths and only occasional demyelinated nerves. (C) The area of demyelinated nerves was significantly less at both day 14 (**P < .001) and day 21 (**P < .001) in rIL-5–treated animals (■) compared with untreated controls (□; n = 10/group). (D) The specificity of the rIL-5 effect was shown by coadministration of rIL-5 and mAb that blocks IL-5 function from day 23 after immunization (gray triangle). This anti–IL-5-blocking mAb abolished the effect of rIL-5 treatment on EAN because the course of EAN in these animals was similar to untreated EAN (○; n = 5/group). rIL-5 treatment from day 13 diminished severity of EAN (●) with significant differences to the rIL-5/anti-IL-5 monoclonal treatment group at day 15 (**P = .004) and day 16 (**P = .003). The rIL-5 CD4⁺CD25⁺ T cells were prepared from these rIL-5–treated and control groups and examined at day 17 after immunization. (E) An increased number of CD4⁺CD25⁺ T cells were observed in peripheral lymphoid tissues of rIL-5–treated animals (■) compared with control EAN (□) and normal Lewis rats (▩; **P < .01). CD4⁺CD25⁺ T cells were taken from animals 4 days after rIL-5 treatment commenced. (F) Enriched CD4⁺CD25⁺ T cells from these rIL-5–treated rats only proliferated to the immunizing Ag PNM and not to unprimed autologous stimulator cells with no Ag or with RTA (***P < .001). Addition of rIL-5 (▩) to culture significantly enhanced proliferation to PNM (***P < .001) compared with cells without rIL-5 that responded to PNM (■). IL-5 had little effect on proliferation of these cells to unprimed autologous stimulators or RTA-primed stimulators. This shows that Tregs were Ag-specific and responded to IL-5.