Immunopathogenesis and nasal tolerance in experimental autoimmune myasthenia gravis

Abstract

Myasthenia gravis (MG) is a classical antibody-mediated autoimmune disease of the neuromuscular junction. Experimental autoimmune myasthenia gravis (EAMG), can be induced in susceptible rat or mouse strains by immunization with acetylcholine receptor (AChR) in complete Freund's adjuvant (CFA). EAMG has many clinical and immunopathological similarities with human MG, thus providing good opportunity to explore the immunopathogenesis of the disease and therapeutic strategies.

To study how age influences the susceptibility to EAMG, adult (10 weeks old) and young (4 weeks old) Lewis rat were immunized with AChR in CFA adjusted to body weight. Young rats were remarkably resistant to EAMG, in comparison with adult rats. This resistance in young rats was associated with lower levels of anti-AChR antibodies and lower levels of AChR induced mRNA expression for both Thl (IFN-y) and Th2 (IL-4, IL-10) cytokines.

Combining in situ tailing and immunocytochemical staining, we observed that infiltrating macrophages in muscle tissue sections during early phase of EAMG in Lewis rats were eliminated by apoptosis at high frequency. These apoptotic macrophages were co-localized in the endplate regions. A transient burst of IL-1ß, IL-6, and TNF-alpha mRNA expressing cells was observed in muscle tissue during the early phase of EAMG in Lewis rats, while numbers of IL-4, IFN-y, cytolysin and TGF-ß were low and no RANTES, MCP, MlP-1alpha and MIP-2 mRNA expressing cells were detected over the course of EAMG.

To evaluate the extent at which humoral immune response against AChR operates in the pathogenesis of EAMG, we immunized B cell knockout (µMT) and wild type C57BL/6 mice with AChR in CFA. µMT mice had no detectable anti-AChR antibodies and remained free from clinical EAMG. The ability of AChR-primed Iymph node cells to proliferate and secrete lFN-y in response to AChR and its dominant myasthenogenic peptide alphal46-162 were intact in µMT as in wild type mice. Similar amount of mRNA for IFN-y, IL4 and IL-10 were detected in AChR primed Iymph node cells in µMT and wild type mice.

We addressed the co-stimulation requirements by investigation of the genesis of EAMG in CD28 and CD40 ligand defident mice (CD28-/-, CD40L-/-). Compared to control C57BL/6 mice, the CD28-/- mice are less susceptible, and CD40L-/- mice are resistant to EAMG. Analysis of T helper functions, reflected by cytokine responses, revealed a switch to a Thl profile in CD28-/ mice. Consistently, levels of AChR-specific IgG antibodies of the IgG1 isotype were decreased in CD28-/- mice. In CD40L-/- mice, both Thl and TH2 cytokine responses were diminished, T cell dependent AChR reactive B cell responses were more severely impaired than in the CD28-/- mice.

To investigate whether nasal administration of AChR modulates ongoing EAMG, Lewis rats were treated nasally with AChR two weeks after immunization with AChR and CFA. Higher amounts of AChR given nasally (600 µg/rat) were required to ameliorate the manifestations of EAMG compared to the amounts necessary for prevention of EAMG. Selective suppression of Thl functions, i.e. IFN-y and IgG2b production, but no influence on Th2 cell functions was observed in the tolerized rats.

Nasal administration of µg doses of AChR + MBP + BPM prior to immunization with a mixture of these antigens + CFA effectively suppressed the incidence and seventy of clinical disease (a combination of EAE, EAMG and EAN), reduced macrophage infiltrations in sections of muscle, spinal cord and sciatic nerve, and down-regulated autoreactive T cell responses to the three autoantigens in Iymphoid organs. A combination of AChR and recombinant rat IFN-y (total 5,000 U/rat) given nasally to Lewis rats before immunization with AChR and CPA reversed the effect of nasal AChR in EAMG rats and escalated T and B cell responses to AChR.

We conclude that (1) Low levels of AChR-reactive Thl and Th2 cells in young Lewis rats are associated with their resistance to EAMG. (2) Apoptosis is a major cause for elimination of infiltrating macrophages during the early phase of EAMG in Lewis rat. (3) Cytokine, but not CC chemokine mRNA expressing cells could be detected in muscle tissues of EAMG in Lewis rat. (4) B cells are critically required for the genesis of clinical EAMG, but not for AChR-specific T cell priming. (5) CD28 and CD40L are differentially required for induction of EAMG. (6) Nasal administration of AChR in ongoing EAMG induced "split tolerance". (7) Nasal tolerance with multiple antigens can prevent more than one experimental autoimmune diseases (EAMG, EAE and EAN). (8) Nasal tolerance can be modulated by nasal administration of minute amounts of IFN7. (9) CD4+ T cells in nasally tolerized rats had decreased LFA-1 expression and upregulated TGF-ß mRNA expression.