J.K. O'Neill

Induction of chronic relapsing experimental allergic encephalomyelitis in Biozzi mice

Experimental allergic encephalomyelitis (EAE) was induced in Biozzi AB/H (antibody high) mice by sensitization with spinal cord homogenate in adjuvant. Biozzi AB/H mice were highly susceptible to EAE induction and followed a chronic relapsing pattern of disease. Disease episodes were characterized by mononuclear infiltration of the central nervous system, with demyelination being particularly evident in relapse. The cellular infiltrates, which were associated with immunoglobulin deposition, consisted of macrophages and primarily CD4-positive T lymphocytes. However, similarly treated Biozzi AB/L (antibody low) mice were markedly less susceptible to EAE induction than AB/H mice. Thus, Biozzi mice should prove valuable for the study of chronic relapsing EAE.

Cytokines in the central nervous system of mice during chronic relapsing experimental allergic encephalomyelitis

Clinical disease phases of chronic relapsing experimental allergic encephalomyelitis (CREAE) in the Biozzi AB/H mouse model are associated with extensive cellular infiltration of the central nervous system, principally the spinal cord. The activation of these cells is further suggested by the immunocytochemical demonstration of cytokines (migration inhibition factor, interferon-gamma, tumour necrosis factor-alpha, and interleukins 1, 2, and 3) within these infiltrates. The in vitro functions attributed to these cytokines indicate their potential role in cell recruitment, activation, and differentiation of the ongoing immune response which could contribute to the pathogenesis of disease.

CD44 is involved in selective leucocyte extravasation during inflammatory central nervous system disease

Clinical signs of experimental autoimmune encephalomyelitis (EAE) are associated with the selective recruitment of CD4+ memory (CD45RBlow CD44high) T cells into the central nervous system (CNS). However, we have found that many of these recently recruited memory cells are CD44low, suggesting that the CD44 antigen may be involved in, and transiently lost during, the extravasation process. Indeed, administration of a CD44‐specific antibody (IM7.8.1) induced leucocyte CD44 shedding and both prevented the development and ameliorated the severity of established EAE by inhibiting mononuclear cell infiltration into the CNS. Trafficking of cells into lymph nodes, however, a property mainly of naïve cells, was essentially unaffected. In contrast, treatment with antibody to very late activation antigen‐4 (VLA‐4) prevented homing to both the CNS and to lymph nodes. This study contests previous reports that dismissed a role for CD44 in inflammation of the CNS and, coupled with observations in murine dermatitis and arthritis, suggests that CD44 is involved in the homing of primed lymphocytes to sites of inflammation. CD44 should therefore be considered a target for immunotherapy of T‐cell‐mediated inflammatory diseases, such as multiple sclerosis.

Mononuclear cell trafficking and plasma protein extravasation into the CNS during chronic relapsing experimental allergic encephalomyelitis in Biozzi AB/H mice

Cellular traffic and plasma protein extravasation across the blood-brain and blood-spinal cord barrier (BBB) have been studied during chronic relapsing experimental allergic encephalomyelitis in Biozzi AB/H mice, using a simultaneous double radioisotope method. There was a general correlation between the clinical course of disease and BBB breakdown, including a resealing of the barrier during remission, although breakdown appeared slightly to precede clinical presentation. The brain was markedly less affected than the spinal cord and was only minimally involved in the relapse phase of disease.

Autoimmune tolerance eliminates relapses but fails to halt progression in a model of multiple sclerosis.

To date there has been poor translation of immunotherapies from rodent models to treatment of progressive multiple sclerosis (MS). In the robust, relapsing Biozzi ABH mouse model of MS, using a combination of a transient deletion of T cells followed by intravenous (i.v.) myelin antigen administration, established relapsing disease in EAE can be effectively silenced. However, when treatment was initiated in late stage chronic-relapsing disease, despite inhibition of further relapses, mice demonstrated evidence of disease progression shown by a deterioration in mobility and development of spasticity and indicates that targeting relapsing, immunological components of MS alone is unlikely to be sufficient to control progression in the late stages of MS.

Suppression of demyelination by mitoxantrone

The mode of action of the immunosuppressant mitoxantrone was examined in murine models of demyelinating disease. The drug has been shown to block antigen induced proliferative activity and to inhibit myelin degradation by leucocytes from paralysed mice. Mitoxantrone blocked myelin breakdown by macrophages although phagocytosis was not affected. Further evidence was obtained to indicate that mitoxantrone acts therapeutically in reducing, or at high dose, preventing signs of EAE developing in mice immunized with spinal cord homogenate and Freunds complete adjuvant. Mitoxantrone also significantly inhibited the incidence of relapse when treatment was initiated during the post-acute remission period.

Local gene therapy with CTLA4-immunoglobulin fusion protein in experimental allergic encephalomyelitis.

It has been reported previously that the induction phase of experimental allergic encephalomyelitis (EAE) is highly sensitive to systemic blockade of stimulation via MHC class II molecules and co‐stimulation via the CD28 : CD80/CD86 pathways. In contrast, the effector phases of EAE were relatively unaffected by similar treatments using MHC class II antigen (Ag)‐specific mAb and cytotoxic T lymphocyte antigen (CTLA)4‐Ig fusion proteins in some studies. This has been attributed to different sensitivities of effector cell function or the poor penetrance of inhibitory proteins into the central nervous system (CNS). To examine this question further, MHC class II Ag‐specific mAb and CTLA4‐Ig were delivered directly into the CNS following EAE induction, and both were found to inhibit disease. While it was found that systemic administration of mouse CTLA4‐Ig could also inhibit the progression of effector immune responses when administered shortly before or during clinical disease, these were significantly more active when delivered directly into the CNS, which probably involved an action on both CD28 ligands, CD80 and CD86. Although mouse CTLA4‐human Ig was therapeutically less efficient than mouse CTLA4‐mouse Ig protein, probably due to the enhanced immunogenicity and lower functional activity, gene delivery of CTLA4‐human Ig into the CNS using a non‐replicating adenoviral vector was more effective than a single injection of CTLA4‐human Ig protein. Gene delivery significantly ameliorated the development of EAE, without necessarily inhibiting unrelated peripheral immune responsiveness. Local gene delivery of CTLA4‐Ig may thus be an important target for immunotherapy of human autoimmune conditions such as multiple sclerosis.

Practical guide to the induction of relapsing progressive experimental autoimmune encephalomyelitis in the Biozzi ABH mouse

Biozzi ABH mice develop a reproducible, relapsing-remitting form of experimental autoimmune encephalomyelitis (EAE) that becomes secondary progressive with disease duration. The relapses observed are T-cell dependent and can be inhibited by immune tolerance induction. In contrast the progressive neurodegeneration is T cell-independent and continues despite the re-induction of immune tolerance. Here we present a practical guide to EAE induction in the ABH mouse and approaches used to control relapses such that both autoimmune-independent and autoimmune-dependent mechanisms of neurodegeneration can be explored. Disease-related weight changes are associated with blood-brain barrier dysfunction and clinical disease. A new method for detecting neurodegeneration is described along with new experimental details that will aid in the undertaking of studies in EAE in mice, with particularly emphasis on ABH mice.

Regulation of chronic relapsing experimental allergic encephalomyelitis by endogenous and exogenous glucocorticoids.

Expression, development and resolution of the acute form of experimental allergic encephalomyelitis (EAE), typically induced in the highly susceptible Lewis rat, are closely regulated by endogenous corticosteroids. Administration of synthetic glucocorticoids also efficiently controls the manifestation of disease. The pivotal role played by the corticosteroids in modifying the induction and progression of EAE is further emphasised by a reversal of corticoid-mediated effects through adrenalectomy or treatment with the steroid receptor antagonist RU486 (mifepristone). Chronic relapsing EAE (CREAE) is characterised by acute symptoms, periods of remission and re-emergence of disease. The mechanisms governing the development of CREAE are unclear, but may require the regulatory influence of endogenous glucocorticoids. The current study has monitored circulating corticosteroids throughout the course of CREAE in the Biozzi ABH mouse and found that major fluctuations in systemic levels coincide with the relapsing-remitting phases of the disease. Furthermore, increasing circulating adrenocorticoids through administration of the steroidal compound dexamethasone markedly suppresses the occurrence of acute signs. The importance of the glucocorticoids in controlling CREAE is again highlighted by the intensification of symptoms and reduction in the survival rate of inoculated mice receiving RU486 prior to and during the acute phase of disease. The data reinforce the amelioratory actions of exogenous and naturally occurring glucocorticoids in the pathogenesis of EAE and extend earlier observations in the monophasic disease by demonstrating corticosteroid-dependent effects in a relapsing-remitting mouse model.

Therapy of chronic relapsing experimental allergic encephalomyelitis and the role of the blood-brain barrier: elucidation by the action of Brequinar sodium

The immunosuppressive effect of the novel 4-quinoline carboxylic acid derivative Brequinar sodium on the chronic relapsing experimental allergic encephalomyelitis CREAE model in the Biozzi AB/H mouse was investigated. Although Brequinar sodium actively inhibited peripheral immune responses, it showed a limited potential to control an ongoing disease of the central nervous system (CNS). Doses of 25 mg/kg inhibited in vivo induced proliferative response and prevented EAE when treated from day 9 post-inoculation (p.i.). However, when administered from day 12 p.i. or during the post-acute remission phase-limited effects on the course of disease were observed. By comparison, treatment with a single high dose of cyclophosphamide (200 mg/kg) at these time points was significantly effective in controlling disease. As a possible explanation of the observed results it is suggested that for a compound to be effective in treating an ongoing immune response in the CNS, it must be capable of crossing the blood-brain barrier and act on the disease-inducing cells activated within the CNS. This hypothesis is supported by the finding that intracerebral injections of Brequinar sodium on day 12 p.i. significantly inhibited disease progression. This suggests that strategies aimed at controlling immune-mediated disease of the CNS require therapeutic doses of the compounds to be delivered into the CNS.