Combined Treatment with Zinc Aspartate and Intravenous Immunoglobulins (IVIGs) Ameliorates Experimental Autoimmune Encephalomyelitis (EAE)

Abstract

Intravenous immunoglobulins (IVIGs) are widely used in replacement therapy of primary and secondary immunodeficiency disorders and in approved autoimmune indications. In addition, IVIG application is used off-label for treatment of other autoimmune diseases, e.g., multiple sclerosis (MS), an inflammatory autoimmune disorder with a clear T cell-mediated immune pathogenesis. The trace element zinc is shown to play a regulatory role in the maintenance of immune functions. Changes of zinc homeostasis affect both the innate and the adaptive immune system. On one hand, therapeutic zinc supplementation can normalize impaired immune functions due to zinc deficiency. On the other hand, therapeutic zinc supplementation is under consideration as a possible option to treat T cell-mediated autoimmune diseases. The aim of the present study was to investigate the influence of IVIG (Octagam®), zinc aspartate (Unizink®), and the combined application of both preparations in the experimental autoimmune encephalomyelitis (EAE), the animal model of MS. Therapeutic intraperitoneal application of zinc aspartate significantly diminished clinical signs during the relapsing-remitting phase of EAE in SJL/J mice. In contrast, IVIG given in a therapeutic manner did not influence the course of EAE. Interestingly, the combined application of both, IVIG and zinc aspartate, significantly reduced the severity of the disease during the acute and the relapsing-remitting phase of the EAE. Our data suggest that the combination of IVIG and zinc aspartate may have beneficial effects in autoimmune diseases, like MS. Further studies should verify the benefit of a controlled immunosuppressive therapy with IVIG and zinc for such diseases.

Figure 1

Effects of preventive and therapeutic application of IVIG on clinical signs of EAE. EAE was induced by immunization of SJL/J mice with PLP (p)139–151 as described in Materials and Methods. Mice were treated i.p. in a preventive manner from day 1 to day 10 (a) or in a therapeutic manner from day 11 to day 19 (b) with 10 mg/day IVIG. PBS treatment served as vehicle control. Treatment periods are indicated by the horizontal bar. Clinical disease scores were recorded daily. Data are presented as daily averages ± SEM of disease scores from 5 mice per treatment group. Significance is indicated by the horizontal bar above the curves. ∗ indicates a significance of p < 0.05 (Mann Whitney analysis).

Figure 2

Effects of therapeutic application of IVIG, zinc aspartate, and the combination of both on clinical signs of EAE. EAE was induced by immunization of SJL/J mice with PLP (p)139–151. Mice were treated i.p. from day 11 to day 15 with 10 mg/day IVIG (a), 30 μg/day zinc aspartate (b), or combination of 10 mg/day IVIG and 30 μg/day zinc aspartate (c). PBS treatment served as vehicle control. Treatment periods are indicated by the horizontal bar. Clinical disease scores were recorded daily. Data are presented as daily averages ± SEM of disease scores from 10 mice per treatment group. Significance is indicated as horizontal bar above the curves. ∗ indicates a significance of p < 0.05 (Mann Whitney analysis).

Figure 3

Effect of therapeutic application of IVIG, zinc aspartate, and the combination of both on formation of inflammatory lesions in the CNS of EAE mice. EAE was induced by immunization of SJL/J mice with PLP_139–151. Mice (n = 4 per group) were treated daily i.p. with 10 mg/day IVIG, 30 μg/day zinc aspartate, the combination of both preparations, or vehicle control from day 11 to day 15. Spinal cords were extracted on day 20, fixed in 4% paraformaldehyde, and embedded in paraffin. Sections were stained with H&E. (a) Representative histology of spinal cord longitudinal sections. Inflammatory infiltrations were visualized by H&E staining. The arrowheads show a typical heavy inflammatory cellular infiltration; original magnification ×200. (b) Inflammatory foci in H&E-stained spinal cord cross sections were quantified. Data represent the mean number of inflammatory foci + SEM; ^∗p < 0.05 (ANOVA).

Figure 4

Effects of combined therapeutic application of IVIG and zinc aspartate on clinical signs of EAE. EAE was induced by immunization of SJL/J mice with PLP (p)139–151. Mice were treated i.p. from day 11 to day 19 with 10 mg/day IVIG and 30 μg/day zinc aspartate. PBS treatment served as vehicle control. Treatment periods are indicated by the horizontal bar. Clinical disease scores were recorded daily. Data are presented as daily averages ± SEM of disease scores from 5 mice per treatment group. Significance is indicated by the horizontal bar above the curves. ∗ indicates a significance of p < 0.05 (Mann Whitney analysis).