Stefanie Gaupp

Selective targeting of regulatory T cells with CD28 superagonists allows effective therapy of experimental autoimmune encephalomyelitis

CD4+CD25+ regulatory T cells (T reg cells) play a key role in controlling autoimmunity and inflammation. Therefore, therapeutic agents that are capable of elevating numbers or increasing effector functions of this T cell subset are highly desirable. In a previous report we showed that a superagonistic monoclonal antibody specific for rat CD28 (JJ316) expands and activates T reg cells in vivo and upon short-term in vitro culture. Here we demonstrate that application of very low dosages of the CD28 superagonist into normal Lewis rats is sufficient to induce T reg cell expansion in vivo without the generalized lymphocytosis observed with high dosages of JJ316. Single i.v. administration of a low dose of the CD28 superagonist into Dark Agouti (DA) rats or Lewis rats that suffered from experimental autoimmune encephalomyelitis (EAE) proved to be highly and equally efficacious as high-dose treatment. Finally, we show that T reg cells that were isolated from CD28-treated animals displayed enhanced suppressive activity toward myelin basic protein–specific T cells in vitro, and, upon adoptive transfer, protected recipients from EAE. Our data indicate that this class of CD28-specific monoclonal antibodies targets CD4+CD25+ T reg cells and provides a novel means for the effective treatment of multiple sclerosis and other autoimmune diseases.

Role of the renin-angiotensin system in autoimmune inflammation of the central nervous system.

Angiotensin II is the principle effector molecule of the renin angiotensin system (RAS). It exerts its various actions on the cardiovascular and renal system, mainly via interaction with the angiotensin II type-1 receptor (AT1R), which contributes to blood pressure regulation and development of hypertension but may also mediate effects on the immune system. Here we study the role of the RAS in myelin-oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis (MOG-EAE), a model mimicking many aspects of multiple sclerosis. Quantitative RT-PCR analyses showed an up-regulation of renin, angiotensin-converting enzyme, as well as AT1R in the inflamed spinal cord and the immune system, including antigen presenting cells (APC). Treatment with the renin inhibitor aliskiren, the angiotensin II converting-enzyme inhibitor enalapril, as well as preventive or therapeutic application of the AT1R antagonist losartan, resulted in a significantly ameliorated course of MOG-EAE. Blockade of AT1R did not directly impact on T-cell responses, but significantly reduced numbers of CD11b+ or CD11c+ APC in immune organs and in the inflamed spinal cord. Additionally, AT1R blockade impaired the expression of CCL2, CCL3, and CXCL10, and reduced CCL2-induced APC migration. Our findings suggest a pivotal role of the RAS in autoimmune inflammation of the central nervous system and identify RAS blockade as a potential new target for multiple sclerosis therapy.

Experimental Autoimmune Encephalomyelitis (EAE) in CCR2−/− Mice: Susceptibility in Multiple Strains

Chemokines are low molecular weight cytokines which act as chemoattractants for infiltrating cells bearing appropriate receptors (CCR) to sites of inflammation. It has been proposed that CCR2 on monocytes is responsible for their recruitment into the central nervous system (CNS) in experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis, and two previous reports have described resistance of CCR2(-/-) mice to EAE. The present study examined three different mouse strains with CCR2 deletions for susceptibility to EAE. Animals were studied up to 4 months post-sensitization and were examined by neuropathology, RNase protection assay, in situ hybridization, and in vitro assays. All three strains were found to be susceptible to EAE: C57BL/6 x J129 and Balb c strains, 100%; and C57BL/6, 67%. Unusual in CNS lesions of CCR2(-/-) mice was an overabundance of neutrophils versus monocytes in wild-type animals. An attempt of the immune system to develop compensatory mechanisms for the lack of CCR2 was evidenced by a corresponding increase in mRNA for other chemokines and CCR. Inasmuch as neutrophils replaced monocytes and led to demyelination, our findings support the concept that promiscuity of chemokines and CCR was able to surmount the deletion of CCR2, still resulting in full expression of this autoimmune disease.

Multiple sclerosis: death receptor expression and oligodendrocyte apoptosis in established lesions.

To determine whether TNF and TRAIL death receptors (DR), and decoy receptors (DcR), play a role in oligodendrocyte depletion in the lesions of chronic multiple sclerosis (MS), we investigated the presence and functionality of these molecules on oligodendrocytes in MS and non-MS brain tissue and on human oligodendrocytes in vitro. For this, we performed immunocytochemistry, Western blotting, TUNEL and FACS analysis for the presence of DR and apoptosis in sections of fresh frozen CNS tissue from cases of chronic MS, other neurologic diseases and normals, and in fetal human oligodendrocytes in vitro. The results showed that although oligodendrocytes demonstrated both DR and DcR, particularly in vitro, there was no predilection of the phenomenon for MS and apoptosis of oligodendrocytes, common in cultures after ligation with TRAIL, was negligible in CNS tissue in situ. Thus, death of oligodendrocytes by apoptosis was an infrequent event in all human CNS samples examined. We postulate that while oligodendrocyte apoptosis might prevail during the initial stages of MS, from our findings other mechanisms probably account for their loss in the established lesion and decoy receptors may play a protective role in oligodendrocyte survival.

MODULATION OF EXPERIMENTAL AUTOIMMUNE NEURITIS IN LEWIS RATS BY ORAL APPLICATION OF MYELIN ANTIGENS

Experimental autoimmune neuritis (EAN) in Lewis rats is a T cell-mediated disease and serves as an animal model of human inflammatory demyelinating neuropathies. EAN can be induced by immunization with complete bovine peripheral nerve myelin (BPM), the myelin protein P2 or its neuritogenic peptide, each emulsified in complete Freunds adjuvant (CFA). The present study evaluates the effect of oral tolerization with BPM or P2 protein on the development of actively induced EAN. Oral administration of BPM strongly suppressed clinical and histological signs of EAN subsequently induced by BPM/CFA, but feeding of P2 protein alone did not affect its course. In contrast, feeding of BPM did not mitigate the course of EAN subsequently induced by immunization with neuritogenic P2 peptide/CFA. Oral therapy with BPM after onset of myelin-induced EAN only slightly ameliorated the further course of disease, but significantly reduced lethality of this severe form of disease. The findings suggest that immunogenicity of the antigens fed determine strength of tolerance, that downregulation of EAN occurs at the site of immunization and not in the nerve, and that active suppression rather than specific anergization is operative in mediating resistance to EAN. However, only partial tolerance to myelin-induced EAN was achieved in naive animals by transfer of spleen/LN cells from rats orally tolerized with BPM. Although methodic factors may have limited the effect of the cells, the result is suggestive of some contribution of anergy to oral tolerance in the present model. Cholera toxin and LPS were identified as oral adjuvants for BPM and prolonged the state of tolerance. However, LPS exhibited proinflammatory properties if EAN was induced early after BPM/LPS-feeding. Thus, oral application of a mixture of myelin components in combination with cholera toxin may be a useful treatment for chronic inflammatory neuropathies considered autoimmune in nature.

Differential efficacy of a synthetic antagonist of VLA-4 during the course of chronic relapsing experimental autoimmune encephalomyelitis

The integrin VLA‐4 has been shown to play a key role in the entry of antigen‐specific T cells into the CNS during autoimmune demyelination. Treatment of animals with experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis, with antibodies to VLA‐4 is known to suppress acute disease. In the present study, a synthetic antagonist of VLA‐4 (TBC 3486) was injected subcutaneously into mice adoptively sensitized for chronic relapsing EAE. TBC 3486 was administered daily for 14 days at early (before acute signs) and late time points (during chronic disease). Early treatment led to marked delay in disease onset and reduction in clinical severity and demyelination. After termination of treatment, clinical severity remained lower than in controls for more than 1 week. TBC 3486‐treated animals showing no clinical signs (at the height of disease in controls) displayed moderate levels of inflammation but little damage to myelin. Late administration of TBC 3486 to animals with chronic EAE had no effect clinically. Immunocytochemistry and Western blotting of CNS tissue from acutely treated animals supported a moderate shift toward a Th2‐type cytokine profile after treatment. Thus, TBC 3486 effectively delayed and reduced the acute (but not chronic) phase of EAE, and this amelioration correlated with changes in the inflammatory molecule profile.

Amelioration of Experimental Autoimmune Encephalomyelitis in IL-4Rα−/− Mice Implicates Compensatory Up-Regulation of Th2-Type Cytokines

The cytokine receptor interleukin (IL)-4R, expressed by lymphocytes, is well known for its role in immunomodulatory signaling and has also been documented on oligodendrocytes, suggesting involvement in glial cell interactions. In the present study, we investigated the clinical course and pathology of experimental autoimmune encephalomyelitis in mice demonstrating deletion of IL-4R and found a correlation with cytokine expression during acute and chronic disease. Wild-type (WT) littermates served as controls. Although IL-4R(-/-) mice displayed a milder course throughout, they showed comparable pathology to WT in the acute phase. However, during the chronic phase, IL-4R(-/-) mice exhibited extensive remyelination and an apparent increase in oligodendrocytes. Cytokine patterns were examined by immunocytochemistry, fluorescence-activated cell sorting, and enzyme-linked immunosorbent assay and were strongly proinflammatory within the central nervous system during the acute phase in WT mice whereas IL-4R(-/-) animals expressed higher levels of IL-6 and IL-10 that became more pronounced with time. The milder experimental autoimmune encephalomyelitis and enhanced remyelination in IL-4R(-/-) mice appeared to be related to a shift toward a Th-2 pattern involving mainly IL-6 and IL-10. These data suggest that IL-4R exerts a negative regulatory role on oligodendrocytes that when deleted results in enhanced myelin repair.

Oral tolerance in experimental autoimmune neuritis (EAN) of the Lewis rat: II. Adjuvant effects and bystander suppression in P2 peptide-induced EAN

Experimental autoimmune neuritis (EAN) in Lewis rats serves as an animal model of human inflammatory demyelinating neuropathies. We previously demonstrated that EAN actively induced by immunization with bovine PNS-myelin can be suppressed by feeding of myelin. This myelin-specific oral tolerance (OT) was enhanced by coapplication of cholera toxin (CT).In the present study, EAN induced by immunization with a neuritogenic P2 peptide was completely prevented by precedent feeding of the peptide. Oral application of the P2 peptide mediated tolerance as efficient as did nasal administration. In contrast to OT by myelin, addition of CT completely prevented oral induction of tolerance to peptide-induced EAN, while adjuvant feeding alone did not modulate disease. As a possible immunological basis to explain the prevention of OT induction by CT, we identified a highly enhanced protein-specific in vitro proliferation of splenocytes from antigen/CT-fed animals compared to those of rats fed with the antigen only. The opposite effects of oral CT in combination with proteins versus myelin confirm our former assumption that CT augments myelin-induced tolerance by its binding to gangliosides present in the myelin but not in the P2 peptide solution, while free CT prevents the induction of OT. It is suggested that CT is a useful adjuvant for OT if coadministered with myelin, but requires chemical linkage to proteins to exert this function. Regulatory lymphocytes orally induced by feeding the protein keyhole limpet hemocyanine (KLH) slightly ameliorated peptide-induced EAN via bystander mechanisms, but injection of KLH in close proximity to the site of peptide-immunization was essential. The restriction of bystander suppression to that site in the lymphoid system where neuritogenic T cells are activated may limit the impact of OT as a bonafide treatment strategy in human autoimmune diseases and underscores the necessity to identify the autoantigens involved.

On the occurrence of hypomyelination in a transgenic mouse model: a consequence of the myelin basic protein promoter?

Abstract Central nervous system hypomyelination is a feature common to anumber of transgenic (Tg) mouse lines that express a variety of unrelated exogenous (i.e. non–central nervous system) transgenes. Inthis report, we document hypomyelination structurally by immunocytochemistry and functionally in the Tg line MBP-JE, which overexpresses the chemokine CCL2 (MCP-1) within oligodendrocytestargeted by a myelin basic protein (MBP) promoter. Analysis ofhypomyelinated optic nerves of Tg mice revealed progressive decrease in oligodendrocyte numbers with age (p < 0.01). Although molecular mechanisms underlying hypomyelination in this and other Tg models remain largely unknown, we present preliminary findings on oligodendrocyte progenitor cell (OPC) cultures in which, although OPC expressed CCR2, the receptor for CCL2, treatment with CCL2 had no significant effect on OPC proliferation, differentiation, or apoptosis. We suggest that hypomyelination in the MBP-JE model might not be due to CCL2 expression but rather the result of transcriptional dysfunction related to random insertion of the MBP promoter that disrupts myelinogenesis and leads to oligodendrocyte demise. Because an MBP promoter is a common denominator in most Tg lines displaying hypomyelination, we hypothesize that use of myelin gene sequences in the regulator region of Tg constructs might underlie thisperturbation of myelination in such models.