Rhonda B. Kean

Uric acid, a peroxynitrite scavenger, inhibits CNS inflammation, blood–CNS barrier permeability changes, and tissue damage in a mouse model of multiple sclerosis

Peroxynitrite (ONOO−), a toxic product of the free radicals nitric oxide and superoxide, has been implicated in the pathogenesis of CNS inflammatory diseases, including multiple sclerosis and its animal correlate experimental autoimmune encephalomyelitis (EAE). In this study we have assessed the mode of action of uric acid (UA), a purine metabolite and ONOO− scavenger, in the treatment of EAE. We show that if administered to mice before the onset of clinical EAE, UA interferes with the invasion of inflammatory cells into the CNS and prevents development of the disease. In mice with active EAE, exogenously administered UA penetrates the already compromised blood‐CNS barrier, blocks ONOO−‐mediated tyrosine nitration and apoptotic cell death in areas of inflammation in spinal cord tissues and promotes recovery of the animals. Moreover, UA treatment suppresses the enhanced blood‐CNS barrier permeability characteristic of EAE. We postulate that UA acts at two levels in EAE: 1) by protecting the integrity of the blood‐CNS barrier from ONOO−‐induced permeability changes such that cell invasion and the resulting pathology is minimized; and 2) through a compromised blood‐CNS barrier, by scavenging the ONOO− directly responsible for CNS tissue damage and death.—Hooper, D. C., Scott, G. S., Zborek, A., Mikheeva, T., Kean, R. B., Koprowski, H., Spitsin, S. V. Uric acid, a peroxynitrite scavenger, inhibits CNS inflammation, blood–CNS barrier permeability changes, and tissue damage in a mouse model of multiple sclerosis. FASEB J. 14, 691–698 (2000)

Regional Differences in Blood-Brain Barrier Permeability Changes and Inflammation in the Apathogenic Clearance of Virus from the Central Nervous System

The loss of blood-brain barrier (BBB) integrity in CNS inflammatory responses triggered by infection and autoimmunity has generally been associated with the development of neurological signs. In the present study, we demonstrate that the clearance of the attenuated rabies virus CVS-F3 from the CNS is an exception; increased BBB permeability and CNS inflammation occurs in the absence of neurological sequelae. We speculate that regionalization of the CNS inflammatory response contributes to its lack of pathogenicity. Despite virus replication and the expression of several chemokines and IL-6 in both regions being similar, the up-regulation of MIP-1β, TNF-α, IFN-γ, and ICAM-1 and the loss of BBB integrity was more extensive in the cerebellum than in the cerebral cortex. The accumulation of CD4- and CD19-positive cells was higher in the cerebellum than the cerebral cortex. Elevated CD19 levels were paralleled by κ-L chain expression levels. The timing of BBB permeability changes, κ-L chain expression in CNS tissues, and Ab production in the periphery suggest that the in situ production of virus-neutralizing Ab may be more important in virus clearance than the infiltration of circulating Ab. The data indicate that, with the possible exception of CD8 T cells, the effectors of rabies virus clearance are more commonly targeted to the cerebellum. This is likely the result of differences in the capacity of the tissues of the cerebellum and cerebral cortex to mediate the events required for BBB permeability changes and cell invasion during virus infection.

The peroxynitrite scavenger uric acid prevents inflammatory cell invasion into the central nervous system in experimental allergic encephalomyelitis through maintenance of blood-central nervous system barrier integrity.

Uric acid (UA), a product of purine metabolism, is a known scavenger of peroxynitrite (ONOO−), which has been implicated in the pathogenesis of multiple sclerosis and experimental allergic encephalomyelitis (EAE). To determine whether the known therapeutic action of UA in EAE is mediated through its capacity to inactivate ONOO− or some other immunoregulatory phenomenon, the effects of UA on Ag presentation, T cell reactivity, Ab production, and evidence of CNS inflammation were assessed. The inclusion of physiological levels of UA in culture effectively inhibited ONOO−-mediated oxidation as well as tyrosine nitration, which has been associated with damage in EAE and multiple sclerosis, but had no inhibitory effect on the T cell-proliferative response to myelin basic protein (MBP) or on APC function. In addition, UA treatment was found to have no notable effect on the development of the immune response to MBP in vivo, as measured by the production of MBP-specific Ab and the induction of MBP-specific T cells. The appearance of cells expressing mRNA for inducible NO synthase in the circulation of MBP-immunized mice was also unaffected by UA treatment. However, in UA-treated animals, the blood-CNS barrier breakdown normally associated with EAE did not occur, and inducible NO synthase-positive cells most often failed to reach CNS tissue. These findings are consistent with the notion that UA is therapeutic in EAE by inactivating ONOO−, or a related molecule, which is produced by activated monocytes and contributes to both enhanced blood-CNS barrier permeability as well as CNS tissue pathology.

Therapeutic intervention in experimental allergic encephalomyelitis by administration of uric acid precursors

Uric acid (UA) is a purine metabolite that selectively inhibits peroxynitrite-mediated reactions implicated in the pathogenesis of multiple sclerosis (MS) and other neurodegenerative diseases. Serum UA levels are inversely associated with the incidence of MS in humans because MS patients have low serum UA levels and individuals with hyperuricemia (gout) rarely develop the disease. Moreover, the administration of UA is therapeutic in experimental allergic encephalomyelitis (EAE), an animal model of MS. Thus, raising serum UA levels in MS patients, by oral administration of a UA precursor such as inosine, may have therapeutic value. We have assessed the effects of inosine, as well as inosinic acid, on parameters relevant to the chemical reactivity of peroxynitrite and the pathogenesis of EAE. Both had no effect on chemical reactions associated with peroxynitrite, such as tyrosine nitration, or on the activation of inflammatory cells in vitro. Moreover, when mice treated with the urate oxidase inhibitor potassium oxonate were fed inosine or inosinic acid, serum UA levels were elevated markedly for a period of hours, whereas only a minor, transient increase in serum inosine was detected. Administration of inosinic acid suppressed the appearance of clinical signs of EAE and promoted recovery from ongoing disease. The therapeutic effect on animals with active EAE was associated with increased UA, but not inosine, levels in CNS tissue. We, therefore, conclude that the mode of action of inosine and inosinic acid in EAE is via their metabolism to UA.

Effective preexposure and postexposure prophylaxis of rabies with a highly attenuated recombinant rabies virus

Rabies remains an important public health problem with more than 95% of all human rabies cases caused by exposure to rabid dogs in areas where effective, inexpensive vaccines are unavailable. Because of their ability to induce strong innate and adaptive immune responses capable of clearing the infection from the CNS after a single immunization, live-attenuated rabies virus (RV) vaccines could be particularly useful not only for the global eradication of canine rabies but also for late-stage rabies postexposure prophylaxis of humans. To overcome concerns regarding the safety of live-attenuated RV vaccines, we developed the highly attenuated triple RV G variant, SPBAANGAS-GAS-GAS. In contrast to most attenuated recombinant RVs generated thus far, SPBAANGAS-GAS-GAS is completely nonpathogenic after intracranial infection of mice that are either developmentally immunocompromised (e.g., 5-day-old mice) or have inherited deficits in immune function (e.g., antibody production or type I IFN signaling), as well as normal adult animals. In addition, SPBAANGAS-GAS-GAS induces immune mechanisms capable of containing a CNS infection with pathogenic RV, thereby preventing lethal rabies encephalopathy. The lack of pathogenicity together with excellent immunogenicity and the capacity to deliver immune effectors to CNS tissues makes SPBAANGAS-GAS-GAS a promising vaccine candidate for both the preexposure and postexposure prophylaxis of rabies.

Comparison of uric acid and ascorbic acid in protection against EAE

Serum levels of uric acid (UA), an inhibitor of peroxynitrite- (ONOO-) related chemical reactions, became elevated approximately 30 million years ago in hominid evolution. During a similar time frame, higher mammals lost the ability to synthesize another important radical scavenger, ascorbic acid (AA), leading to the suggestion that UA may have replaced AA as an antioxidant. However, in vivo treatment with AA does not protect against the development of experimental allergic encephalomyelitis (EAE), a disease that has been associated with the activity of ONOO- and is inhibited by UA. When compared in vitro, UA and AA were found to have similar capacities to inhibit the nitrating properties of ONOO-. However UA and AA had different capacities to prevent ONOO- -mediated oxidation, especially in the presence of iron ion (Fe3+). While UA at physiological concentrations effectively blocked dihydrorhodamine-123 oxidation in the presence of Fe3+, AA did not, regardless of whether the source of ONOO- was synthetic ONOO-, SIN-1, or RAW 264.7 cells. AA also potentiated lipid peroxidation in vivo and in vitro. In conclusion, the superior protective properties of UA in EAE may be related to its ability to neutralize the oxidative properties of ONOO- in the presence of free iron ions.

Loss of blood–brain barrier integrity in the spinal cord is common to experimental allergic encephalomyelitis in knockout mouse models

Experimental allergic encephalomyelitis (EAE) is an inflammatory demyelinating disease of the CNS that is used to model certain parameters of multiple sclerosis. To establish the relative contributions of T cell reactivity, the loss of blood–brain barrier (BBB) integrity, CNS inflammation, and lesion formation toward the pathogenesis of EAE, we assessed the incidence of EAE and these parameters in mice lacking NF-κB, TNF-α, IFN-αβ receptors, IFN-γ receptors, and inducible nitric oxide synthase. Although increased myelin oligodendrocyte glycoprotein-specific T cell reactivity was generally associated with a more rapid onset or increased disease severity, the loss of BBB integrity and cell accumulation in spinal cord tissues was invariably associated with the development of neurological disease signs. Histological and real-time RT-PCR analyses revealed differences in the nature of immune/inflammatory cell accumulation in the spinal cord tissues of the different mouse strains. On the other hand, disease severity during the acute phase of EAE directly correlated with the extent of BBB permeability. Thus, the loss of BBB integrity seems to be a requisite event in the development of EAE and can occur in the absence of important inflammatory mediators.

The development of monoclonal human rabies virus-neutralizing antibodies as a substitute for pooled human immune globulin in the prophylactic treatment of rabies virus exposure.

To provide a more defined and safer replacement for the human rabies immune globulin (HRIG) from pooled serum which is currently used for treatment of exposure to rabies virus we have developed a series of human rabies virus-specific monoclonal antibodies. Mouse-human heterohybrid myeloma cells producing rabies virus-specific human monoclonal antibodies were prepared using B cells obtained from volunteers recently-immunized with a commercial rabies virus vaccine (HDCV). Cell lines producing antibody which neutralized the Evelyn-Rokitnicki-Abelseth (ERA) rabies virus strain in vitro were cloned and the resulting monoclonal antibodies characterized for isotype, specificity against a variety of rabies virus isolates, and neutralization capacity. The ability of the monoclonal antibodies to neutralize a variety of rabies virus strains in vitro correlated with their binding specificity for these viruses in an enzyme-linked immunoadsorbant assay (ELISA). A number of these antibodies have proven suitable for the formulation of a prophylactic human monoclonal antibody-based reagent which would provide significant advantages to the HRIG in having defined, reproducible specificity, lessened possibility of contamination with viral pathogens, and consistent availability.

A Peroxynitrite-Dependent Pathway Is Responsible for Blood-Brain Barrier Permeability Changes during a Central Nervous System Inflammatory Response: TNF-α Is Neither Necessary nor Sufficient

Elevated blood-brain barrier (BBB) permeability is associated with both the protective and pathological invasion of immune and inflammatory cells into CNS tissues. Although a variety of processes have been implicated in the changes at the BBB that result in the loss of integrity, there has been no consensus as to their induction. TNF-α has often been proposed to be responsible for increased BBB permeability but there is accumulating evidence that peroxynitrite (ONOO−)-dependent radicals may be the direct trigger. We demonstrate here that enhanced BBB permeability in mice, whether associated with rabies virus (RV) clearance or CNS autoimmunity, is unaltered in the absence of TNF-α. Moreover, the induction of TNF-α expression in CNS tissues by RV infection has no impact on BBB integrity in the absence of T cells. CD4 T cells are required to enhance BBB permeability in response to the CNS infection whereas CD8 T cells and B cells are not. Like CNS autoimmunity, elevated BBB permeability in response to RV infection is evidently mediated by ONOO−. However, as opposed to the invading cells producing ONOO− that have been implicated in the pathogenesis of CNS inflammation, during virus clearance ONOO− is produced without pathological sequelae by IFN-γ-stimulated neurovascular endothelial cells.

The Central Nervous System Inflammatory Response to Neurotropic Virus Infection Is Peroxynitrite Dependent

We have recently demonstrated that increased blood-CNS barrier permeability and CNS inflammation in a conventional mouse model of experimental allergic encephalomyelitis are dependent upon the production of peroxynitrite (ONOO−), a product of the free radicals NO· and superoxide (O2·−). To determine whether this is a reflection of the physiological contribution of ONOO− to an immune response against a neurotropic pathogen, we have assessed the effects on adult rats acutely infected with Borna disease virus (BDV) of administration of uric acid (UA), an inhibitor of select chemical reactions associated with ONOO−. The pathogenesis of acute Borna disease in immunocompetent adult rats results from the immune response to the neurotropic BDV, rather than the direct effects of BDV infection of neurons. An important stage in the BDV-specific neuroimmune response is the invasion of inflammatory cells into the CNS. UA treatment inhibited the onset of clinical disease, and prevented the elevated blood-brain barrier permeability as well as CNS inflammation seen in control-treated BDV-infected rats. The replication and spread of BDV in the CNS were unchanged by the administration of UA, and only minimal effects on the immune response to BDV Ags were observed. These results indicate that the CNS inflammatory response to neurotropic virus infection is likely to be dependent upon the activity of ONOO− or its products on the blood-brain barrier.