Barbara Cannella

Identification of autoantibodies associated with myelin damage in multiple sclerosis

The molecular mechanisms underlying myelin sheath destruction in multiple sclerosis lesions remain unresolved. With immunogold–labeled peptides of myelin antigens and high–resolution microscopy, techniques that can detect antigen–specific antibodies in situ, we have identified autoantibodies specific for the central nervous system myelin antigen myelin/oligodendrocyte glycoprotein. These autoantibodies were specifically bound to disintegrating myelin around axons in lesions of acute multiple sclerosis and the marmoset model of allergic encephalomyelitis. These findings represent direct evidence that autoantibodies against a specific myelin protein mediate target membrane damage in central nervous system demyelinating disease.

Demyelination in primate autoimmune encephalomyelitis and acute multiple sclerosis lesions: A case for antigen-specific antibody mediation

Neuropathological and ultrastructural features of central nervous system demyelination were compared in marmoset experimental autoimmune encephalomyelitis (EAE) induced with myelin/oligodendrocyte glycoprotein (MOG), and in 3 cases of multiple sclerosis (MS) displaying recent lesions. At the edges of EAE and MS lesions, a zone of myelin vacuolation was common, whereas in the lesion proper, myelin sheaths were consistently transformed into vesiculated membranous networks. These networks became dissociated from axons by cell processes from macrophages. Oligodendrocytes were remarkably spared and evidence of myelin repair was present but not prominent. Axonal pathology was more common in the MS material than in marmoset EAE. Immunocytochemistry, using gold‐labeled encephalitogenic peptides of MOG and silver enhancement to detect MOG autoantibodies, revealed the presence of MOG‐specific autoantibodies over vesiculated myelin networks. Gold‐labeled antibody to IgG also gave a positive reaction. Gold‐labeled peptide of myelin basic protein did not react with MOG/EAE tissue, but the same conjugate gave positive staining in MS (and in marmoset EAE induced by whole white matter), perhaps indicating broader spectrum immunoreactivity or sensitization to myelin antigens. Thus, vesicular disruption of myelin was a constant feature in these evolving, highly active lesions in primate EAE and MS and appeared causally related to the deposition of antigen‐specific autoantibodies. Ann Neurol 1999;46:144–160

IL-10 FAILS TO ABROGATE EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS

Mice adoptively‐sensitized to develop chronic relapsing experimental autoimmune encephalomyelitis (EAE), a model for the human demyelinating condition, multiple sclerosis (MS), were given injections of recombinant human IL‐10 at various timepoints post‐sensitization in an attempt to abrogate disease development. IL‐10 is a Th2 immunomodulatory cytokine with known down‐regulatory effects upon Th1 responses and macrophages. Contrary to a previous report on EAE and the predicted outcome, after repeated experiments, IL‐10 was found to elicit a worsening or no effect upon EAE in the mouse. Animals were studied clinically, histopathologically and immunocytochemically. On no occasion was disease ameliorated by IL‐10. Pretreatment with IL‐10 of lymph node cells used to transfer EAE had no effect upon disease outcome, indicating that the cells were already committed effectors. Administration of anti‐IL‐10 monoclonal antibody before onset of signs had no effect when given early post‐sensitization and caused marked worsening when given immediately before onset of signs. In the context of this autoimmune demyelinating model, these results suggest that IL‐10 alone is insufficient to reverse the effector response and indeed may serve to enhance the cascade of events in EAE.

Activation of NF-κB and c-jun Transcription Factors in Multiple Sclerosis Lesions: Implications for Oligodendrocyte Pathology

Oligodendrocytes are a major target of the purported autoimmune response in multiple sclerosis (MS) lesions, but little is known about the mechanisms underlying their demise. Despite the expression of proapoptotic receptors, these cells are rarely seen to undergo apoptosis in situ. On the other hand, cytotoxic mediators present in MS lesions, such as tumor necrosis factor-alpha, are known to generate survival signals through the activation of the transcription factors NF-kappaB and c-jun. The aim of this study was to investigate in chronic active and silent MS lesions and control white matter the expression of c-jun, its activating molecule, JNK, as well as NF-kappaB complex and its inhibitor, IkappaB. By immunohistochemistry we found negligible reactivity for these molecules in control white matter and silent MS plaques. In active MS lesions, double-label immunohistochemistry with oligodendrocyte markers showed up-regulation of the nuclear staining for both NF-kappaB and JNK on a large proportion of oligodendrocytes located at the edge of active lesions and on microglia/macrophages throughout plaques. Oligodendrocytes showed no reactivity for IkappaB, which was predominantly confined to the cytoplasm of microglia/macrophages. We hypothesize that activation of these transcriptional pathways may be one mechanism accounting for the paucity of oligodendrocyte apoptosis reported in MS.

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: Cytokine receptors on oligodendrocytes predict innate regulation

Multiple sclerosis (MS) is an immune‐mediated demyelinating condition in which numerous soluble mediators have been implicated. We have extended the repertoire of cytokines studied in MS tissue by examining interleukin (IL‐4), IL‐6, IL‐10, IL‐12, IL‐18, interferon (IFNγ), and their receptors and have compared patterns with those seen in normal subjects and other neurological diseases (OND). Expression was evaluated by immunocytochemistry and Western blots. Remarkably, oligodendrocytes expressed all the cytokine receptors examined, particularly Th2‐type, constitutively in normal subjects and upregulated in disease. Microglial cells also expressed cytokine receptors at similar levels. Cytokine expression was invariably a feature of microglial cells, except for IL‐10, which was exclusively astrocytic. Oligodendrocytes did not display cytokines, except for low levels of IL‐18. Although no pattern was specific for MS, most molecules were upregulated in MS and OND. Downstream JAK/STAT molecules were correspondingly upregulated. Cytokine receptors on oligodendrocytes (and microglia), and their corresponding ligands on microglia (and astrocytes), may implicate paracrine/autocrine regulation and may bespeak innate immunity in the central nervous system.

Therapeutic potential of phosphodiesterase type 4 inhibition in chronic autoimmune demyelinating disease

It was recently demonstrated that selective phosphodiesterase type 4 (PDE4) inhibition suppresses the clinical manifestations of acute experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), and inhibits the production of tumor necrosis factor-alpha (TNF-alpha), a pathogenetically central cytokine. Since the most common presentation of MS in humans is a relapsing-remitting course, we investigated the therapeutic potential of PDE4 inhibition in the relapsing-remitting EAE model of the SJL mouse. Administration of rolipram, the prototypic PDE4 inhibitor, reduced the clinical signs of EAE during both the initial episode of disease and subsequent relapses. In parallel, there was marked reduction of demyelination and also less inflammation throughout the central nervous system (CNS) of rolipram-treated animals. Gene expression of proinflammatory cytokines in the CNS was reduced in most of the rolipram-treated animals. Additional experiments demonstrated that PDE4 inhibition acted principally by inhibiting the secretion of Th1 cytokines, however, the encephalitogenic potential of myelin basic protein-specific T cells was not impaired. Our findings suggest that PDE4 inhibitors are a promising cytokine-directed therapy in chronic demyelinating disease.

Long-term treatment of chronic relapsing experimental allergic encephalomyelitis by transforming growth factor-β2

It had been demonstrated previously that the administration of transforming growth factor-beta 1 (TGF-beta 1) reduced the clinical severity of experimental allergic encephalomyelitis (EAE). Treatment with the related immunosuppressive molecule, TGF-beta 2, resulted in similar inhibition of T cell activation and proliferation in vitro. Long-term treatment was effective in reducing clinical severity of EAE and the number of relapses in mice receiving either myelin basic protein- or peptide-91-103-specific T cell lines. When examined histologically, mice that had received TGF-beta 2 demonstrated significantly less inflammation and demyelination in the central nervous system. Examination of other organs demonstrated no pathology or deleterious side effects from long-term TGF-beta 2 therapy. These findings have relevance for the use of TGF-beta 2 as a therapeutic agent for the human demyelinating disease, multiple sclerosis.

Neuregulin and erbB receptor expression in normal and diseased human white matter.

Human white matter from non-neurologic cases, multiple sclerosis (MS) and other neurologic diseases (OND, inflammatory and non-inflammatory), was subjected to immunocytochemistry and Western blotting for expression of the neuregulin, glial growth factor-2 (GGF2), and its receptors, erbB2, erbB3 and erbB4. GGF2 has previously been shown to have mitogenic effects upon oligodendrocytes in vitro and an enhancing effect upon remyelination in animals with autoimmune demyelination. In all types of human white matter examined, expression of the ligand GGF2 and its three receptors was consistently found on oligodendrocytes, with higher levels being seen in cases of MS. Expression was also seen, albeit at lower levels, on astrocytes and microglial cells, the latter most commonly in MS and OND. In human lymph node tissue, some lymphocytes were positive for erbB2, erbB3 and erbB4. Western blots confirmed the presence of all three receptors in normal, MS and OND white matter. GGF2 and erbB receptor expression did not correlate with areas of remyelination and reactivity occurred throughout the tissue, with some increase in intensity at the edge of MS lesions. Examination of precursor oligodendrocyte immunoreactivity (with anti-PDGF-Ralpha and NG2), revealed widespread expression throughout both normal and diseased white matter. The presence of GGF2 and its receptors on oligodendrocytes and lymphocytes render this cell type a candidate for functional signaling via this pathway, perhaps in relationship to myelinating activity.

Insulin-Like Growth Factor-1 Fails to Enhance Central Nervous System Myelin Repair during Autoimmune Demyelination

Previous studies have shown that insulin-like growth factor-1 (IGF-1) has beneficial effects, both clinically and histopathologically, on experimental autoimmune encephalomyelitis (EAE), although results vary depending on species and treatment regimen. The present study investigated whether IGF-1, delivered at different time points during the acute and chronic phases of adoptively transferred EAE in SJL mice, had the ability to affect or enhance myelin regeneration. Central nervous system tissue sampled at different stages of treatment was subjected to detailed neuropathological, immunocytochemical and molecular analysis. The results revealed some transient clinical amelioration and low level remyelination after IGF-1 administration during the acute phase of EAE. However, central nervous system tissue from acute phase treated animals sampled at chronic time points and from animals given IGF-1 during the chronic phase revealed no enhancing effect on remyelination in comparison to vehicle-treated controls. Examination of oligodendrocyte progenitor populations also revealed no differences between IGF-1- and vehicle-treated groups. At the cytokine level, the immunomodulatory molecules TGF-beta2 and TGF-beta3 displayed significant decreases that may have contributed to the transient nature of the effect of IGF-1 on EAE. Together with evidence from previous studies, it appears doubtful that IGF-1 is a good candidate for treatment in multiple sclerosis, for which EAE serves as a major model.