in C57BL/6 Mice of Chronic Autoimmune Myasthenia Gravis Absence of IL-4 Facilitates the Development

Myasthenia gravis (MG) is a T cell-dependent, Ab-mediated autoimmune disease. Ab against muscle acetylcholine receptor (AChR) cause the muscular weakness that characterizes MG and its animal model, experimental MG (EMG). EMG is induced in C57BL6 (B6) mice by three injections of Torpedo AChR (TAChR) in adjuvant. B6 mice develop anti-TAChR Ab that cross-react with mouse muscle AChR, but their CD4 (cid:1) T cells do not cross-react with mouse AChR sequences. Moreover, murine EMG is not self-maintaining as is human MG, and it has limited duration. Several studies suggest that IL-4 has a protecting function in EMG. Here we show that B6 mice genetically deﬁcient in IL-4 (IL-4 (cid:2) / (cid:2) ) develop long-lasting muscle weakness after a single immunization with TAChR. They develop chronic self-reactive Ab, and their CD4 (cid:1) T cells respond not only to the TAChR and TAChR (cid:3) subunit peptides, but also to several mouse AChR (cid:3) subunit peptides. These results suggest that in B6 mice, regulatory mechanisms that involve IL-4 contribute to preventing the development of a chronic Ab-mediated autoimmune response to the AChR. The Journal of Immunology, 2003, 170: 604–612. (cid:2) -bungarotoxin ( 125 I-labeled (cid:2) BTX) and a polyclonal Ab against Ig. We measured the concentration of Ab that cross-reacted with mouse muscle AChR by a modiﬁcation of the anti-TAChR Ab assay, using a Triton X-100 extract of muscle from naive B6 mice as the source of AChR, at a concentration of (cid:3) 1 nM (cid:2) BTX binding sites, labeled by overnight incubation with 125 I-labeled (cid:2) BTX (10). We each serum set up precipitation using After All concentrations are as trations of precipitated 125 I-labeled (cid:2) BTX binding sites. We used either the total splenocytes or the splenocytes depleted in CD8 (cid:1) cells (CD4 (cid:1) splenocytes) using rat anti-mouse CD8 (cid:1) Ab (BD PharMingen) and paramagnetic beads covalently attached to goat anti- rat IgG (Polyscience, Warrington, PA). We tested the proliferative response of cells using triplicate or quadruplicate wells (100,000–200,000 cells/well) and the appropriate selection among the following stimulants: PHA (10 (cid:3) g/ml; Sigma-Aldrich, St. Louis, MO), TAChR (0.5–5 (cid:3) g/ml), and individual TAChR or mouse AChR (cid:2) subunit peptides (10 (cid:3) g/ml). Two sets of control wells were cultured without any Ag or with a 20- residue peptide synthesized by the same method as the AChR peptides and unrelated to the AChR sequence. We determined the rate of cell prolifer- ation from the incorporation of [ 3 H]thymidine, measured by liquid scintil-lation. We assessed the signiﬁcance of the difference of the average [ 3 H]thymidine incorporation of cultures stimulated with a given Ag and that of the control wells, using two-tailed Student’s t test and the program Excel (Microsoft Corp., Redmond, WA). When an Ag induced a signiﬁcant ( p (cid:5) 0.05) increase in cell proliferation, we calculated the stimulation index (SI; the ratio between the cpm of a culture in the presence of the Ag and the average basal proliferation of the same cells). The use of SI nor- malized results and allowed comparison of experiments conducted at different times, with different mice. splenocytes from TAChR-immunized IL-4 (cid:2) / (cid:2) and WT mice, euthanized 2 or 4 wk after the immunization. We cultured the cells in 96-well ELISPOT plates (300,000 cells/well) coated with anti-mouse IFN- (cid:5) mAb (BD PharMingen), and stimulated them with 2.5 (cid:3) g/ml of TAChR for 48 h. We used a biotin-conjugated secondary Ab and streptavidin-conjugated HRP (Vector Laboratories, Burlingame, CA) as the revealing system. The development solution contained 800 (cid:3) l of 100 mg of 3-amino-9-ethyl carbazole in 10 ml of N , N -dimethylformamide, 24 ml of 0.2 M acetic acid, 0.2 M sodium acetate (pH 5.0), and 12 (cid:3) l of 30% H 2 O 2 . We also determined the concentration of IFN- (cid:5) in the supernatant of CD4 (cid:1) splenocytes from TAChR-immunized IL-4 (cid:2) / (cid:2) and WT mice euthanized 4 wk after the immunization, cultured for 48 h in the presence or the absence of 2.5 (cid:3) g/ml of TAChR. /