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.
The chronic autoimmune disease multiple sclerosis (MS) is the most frequent demyelinating disease of the central nervous system (CNS) with a prevalence of 0.1% in Northern America and Europe. MS can affect all functional systems of the CNS, leading to symptoms like weakness of one or several limbs, optic neuritis, cerebellar or brainstem dysfunction, sensory deficits, and cognitive impairment [
The trace element zinc is shown to be essential for a wide range of physiological processes, including cell and tissue differentiation, proliferation, and apoptosis. Zinc is involved in the regulation of numerous structural and catalytic functions, in protein-protein interactions, and in signal transduction of several cell types [
Based on these observations, therapeutic zinc supplementation is considered for a long time as a possible option for T cell-mediated autoimmunity [
Intravenous immunoglobulin (IVIG) preparations contain pooled immunoglobulin G (IgG) from the plasma of approximately thousand blood donors. IVIGs are used in a variety of conditions, especially in replacement therapy of primary and secondary immunodeficiency disorders, in approved autoimmune diseases, and in off-label indications for several autoimmune diseases [
Concerning the combined application of IVIG and zinc preparations in EAE, no studies exist as yet. Thus, we wanted to answer the question whether the combination of IVIG and zinc aspartate is effective in EAE as an animal model of MS.
IVIG (Octagam®) was purchased from Octapharma GmbH (Langenfeld, Germany) and zinc aspartate (Unizink®) from Köhler Pharma GmbH (Alsbach-Hähnlein, Germany). Proteolipid protein (PLP) peptide (p)139–151, corresponding to the mouse sequence (HSLGKWLGHPDKF) was synthesized on a peptide synthesizer and purified by HPLC.
Female SJL/J mice, age 10–12 weeks, were purchased from JANVIER LABS (Le Genest-Saint-Isle, France) and housed in the animal facilities of the medical faculty of the Otto-von-Guericke-University, Magdeburg. All procedures were conducted according to protocols approved by the Institutional Animal Care and Use Committee (number 42502-2-781 UniMD).
Female SJL/J mice were immunized subcutaneously (s.c.) in depots distributed over 4 spots across the flanks with 200
For therapeutic treatment, the mice received IVIG (10 mg/day), 30
The mice were scored daily for clinical signs of EAE according to the following increasing severity scale: 0: no disease; 1: tail weakness (tail plegia); 2: hindlimb paraparesis and/or weak righting reflex; 3: hindlimb paraplegia; 4: paraplegia with forelimb weakness or paralysis; and 5: moribund animals. Mice with intermediate clinical signs were scored in 0.5 increments. For reasons of animal welfare, the mice were killed when reaching a score of 3 or above. Daily clinical scores were calculated as the average of all individual disease scores of each group [
For histological analysis, the mice were euthanized at day 20 postimmunization. Spinal cords were removed, fixed in 4% paraformaldehyde, embedded in paraffin, and stained with hematoxylin and eosin (H&E). Thoracic and lumbar spinal cord sections were evaluated, and total numbers of inflammatory foci were determined by an examiner blinded to the treatment status of the animal.
Statistical comparison of EAE disease severity was accomplished by performing a Mann Whitney analysis as described previously [
Recently, we could show that i.p. administration of 30
In order to characterize the effect of IVIG application in active EAE, SJL/J mice were immunized with PLP (p)139–151. IVIGs (10 mg/day) were given i.p. in a preventive (treated from day 1 to day 10 after immunization) or in a therapeutic manner (treated from day 11 to day 19). PBS treatment served as vehicle control. We observed that the IVIG preparation was capable of diminishing the severity of EAE only in a preventive application (Figure
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.
Next, we wanted to answer the question whether the therapeutic application of IVIG in combination with zinc aspartate can diminish the severity of the clinical course of EAE. The mice were treated i.p. with 10 mg/day IVIG, 30
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
As expected, therapeutic IVIG application alone from day 11 to day 15 after immunization showed no positive effect on the severity of the EAE (Figure
This observation indicates a synergistic effect of IVIG and zinc in the treatment of relapsing-remitting EAE in mice.
Confirming the clinical data, histopathological analysis of EAE spinal cord tissues showed significantly decreased numbers of total inflammatory foci in EAE mice, which were treated i.p. with zinc aspartate or with the combination of zinc aspartate and IVIG from day 11 to day 15 (Figure
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 PLP139–151. Mice (
Since therapy over a time period of 5 days already showed a significant improvement of EAE severity, we next investigated the combined effect of IVIG and zinc aspartate in a prolonged treatment protocol from day 11 to day 19 after immunization (Figure
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
In the past twenty years, it has been demonstrated that zinc is a key regulator of the immune system, capable of stimulating or suppressing different immune cells
Changes of zinc homeostasis affect both the innate and the adaptive immune system. On one site, impaired immune function due to zinc deficiency can be normalized by therapeutic zinc supplementation [
In the absence of other therapeutic options, IVIG is used as part of an “off-label therapy” in several autoimmune diseases and, e.g., for the postpartum prophylaxis of MS in pregnant women [
Comparing the effect of prophylactic and therapeutic IVIG administration on the course of active EAE in SJL/J mice, we found that IVIGs are capable of diminishing the severity of EAE in this animal model only after preventive application. This is in line with studies by other authors [
So far, no studies exist concerning the combined application of IVIG and zinc preparations in EAE. Thus, in the present study, we asked the question whether the combination of IVIG and zinc aspartate is effective in active EAE in SJL/J mice. Therefore, we combined the application of IVIG and zinc aspartate in a therapeutic manner and observed a significant reduction of the severity of the disease relapses. The therapeutic treatment with IVIG showed no effect on the acute phase of EAE or the further course. The therapeutic administration of zinc prevented the first relapse of the EAE but had no significant effect on the acute phase of the disease. Interestingly, the combined application of IVIG and zinc caused a rapid remission of the acute phase of EAE and prevented further relapses. Only about 100 days after the therapy, the course of disease of treated mice matched the course of the control group. This therapeutic effect not only was clinically evident from the EAE score but also could be demonstrated histologically. The number of inflammatory foci in the spinal cord was significantly reduced in animals treated with zinc aspartate alone or with the combination of IVIG and zinc compared to the control group. Further studies should be performed to investigate the effect of the combined treatment of zinc aspartate and IVIG after the first and/or second relapse of EAE.
For both MS and EAE, a T cell-mediated pathogenesis, in which Th1 and Th17 cells play a crucial role, is discussed [
Recently, it was demonstrated in the EAE animal model that IVIG can inhibit the initiation of pathogenic immune response by inhibiting the polarization of naïve T cells into Th17 and Th1 cells, simultaneously increasing regulatory T (Treg) cells [
Furthermore, it is known that the immunosuppressive cytokine TGF-
In order to perform its function, latent TGF-
The present work shows a significant improvement of the clinical course of EAE under zinc or zinc and IVIG administration. The results suggest that the combination of zinc and IVIG is capable of inhibiting the proliferation of Th1 and Th17 cells and thus suppressing the CNS inflammatory response during EAE. In addition, it was shown that the combination of IVIG and zinc significantly reduces the severity of the EAE in comparison to the administration of zinc aspartate alone.
In addition to Th1 and Th17 cells, CD4+CD25+FoxP3+ Treg cells play also an important role in the pathogenesis of EAE and MS [
Complete Freund’s adjuvant
Central nervous system
Experimental autoimmune encephalomyelitis
Hematoxylin and eosin
High-performance liquid chromatography
Interleukin
Interferon
Immunoglobulin G
Intraperitoneal
Intravenous immunoglobulins
Multiple sclerosis
Phosphate-buffered saline
Peripheral blood mononuclear cells
Proteolipid protein
Pertussis toxin
Transforming growth factor-
Regulatory T cells.
The data used to support the findings of this study are included within the article.
There is no conflict of interest in this study.
We acknowledge grant support by the research alliance “Autonomy in Old Age” (EFRE and the State of Saxony-Anhalt). The authors thank Mrs. Marita Lotzing for the excellent technical assistance.