Immune Mechanisms in Viral Encephalomyelitis

Immune Mechanisms in Viral Encephalomyelitis Abstract In susceptible strains of mice, a viral infection caused by the virus “Theiler’s murine encephalomyelitis (TMEV),” causes a biphasic disease of the nervous system. This virus may serve as a useful animal model of major human nervous system diseases. Prior studies of this viral infection in susceptible mice revealed during the first month after intracranial injection of TMEV, the disease is similar to polio, but in a later stage the mice develop demyelination which resembles multiple sclerosis. This present study evaluated the role of the immune system in such infections. Specifically, the helper T cells (L3T4 or CD4+ T cells) were depleted by injection of rat monoclonal antibodies prior to infection with the virus. Mice with such T-cell depletion were unable to develop antibodies against TMEV and thus succumbed rapidly to the infection, dying within one month. In other mice, T cells were depleted after infection but before the demyelination stage. In these mice, the viral-induced paralysis was reduced from 77% to 36% of infected mice. In normal mice, not depleted of T cells, TMEV infection stimulated antibody production against myelin, suggesting that demyelination might be an autoimmune disease. Introduction According to a variety of cited previous publications, the authors summarize what is known about Theiler’s murine encephalomyelitis virus (TMEV). This virus infects mice and has many characteristics similar to human polio virus. In mice TMEV causes a mild intestinal infection and sometimes paralysis. One particular genetic strain of this virus (TO) causes a biphasic encephalomyelitis. In susceptible mice, there is an early stage (up to the first 30 days after intracranial injection) mice exhibit poliolike symptoms, presumably by killing some spinal motor neurons. Later, the disease appears more like human multiple sclerosis, causing inflammatory demyelinating lesions. Prior studies had shown that such demyelination could be prevented by blocking the immune response. T cells have been implicated in delayed hypersensitivity conditions that had been reported to correlate with demyelination . The investigators of the study reasoned that T cells might be involved in TMEV infections. Thus, they used a previously known system that could block T-cell responses. Monoclonal antibodies from rats 1 are known to deplete certain T cells and thus interfere with their immune functions. Here, the course of infection in TMEV susceptible mice was tracked in mice whose T cells (a sub-population known as L3T4-positive T cells) were depleted by exposure to the monoclonal antibodies. Methods Animals. The study used four- to five-week old female mice of the CBA strain (known to be susceptible to TMEV). The virus. The virus supplied from another laboratory, which had grown it in cell culture. The viruscontaining supernatant from the culture was stored at -70o C. Assay of virus in infected mice. Brains and spinal cord were removed from infected mice and stored in special media (Glasgow’s modified Eagle media: contained antibiotic, fungicide, buffer solution, glutamine,). The tissue was then homogenized and centrifuged to separate out the virus in the supernatant. Preparation of Monoclonal Antibody Against T Cells. Antibody was supplied by a collaborating laboratory. The preparation process involved a cell culture system in which a rat tumor culture (myeloma cells) are fused to spleen cells from animals immunized with mouse T cells. The resulting fusion partners secrete pure homogenous antibodies against mouse T cells. Antibody Measurement. Partially purified TMEV was used in an ELISA test to measure monoclonal antibody concentrations. Myelin Preparation and Detection of Antibodies in Myelin. Myelin from SJL mouse-strain brain was extracted and purified by a generally accepted sucrose gradient separation process. The myelin was then used in an ELISA test for antibodies. T-cell Depletion: CBA strain mice, with thymus gland removed, were injected with 0.1 ml of monoclonal antibody (10 mg/ml) at either one day prior to TMEV infection or six days afterwards. A fluorescence-activated cell sorting indicated that this process killed 98% of T cells. For the mice that were used to study the demyelinating phase of infection, similar T-cell depletion was produced at 65 or 72 days after infection with TMEV. Virus Injection: At day 0, mice were anesthetized with ether, and injected intracranially in the right hemisphere with 20 microliters of media solution containing the TMEV virus. Other control mice were similarly injected, but with physiological saline. Evaluation of Clinical Signs of Demyelination: Investigators performed a behavioral assay twice weekly without knowing which treatment group each mouse was in. The test involved scoring of a paw grasp response when mice were placed on a wire mesh, ability to accomplish the righting reflex, and signs of incoordination and/or incontinence. Histological Examination of Spinal Cords. At the end of the experiment, mice were anesthetized again with ether and perfused through the heart with a chemical fixative (4% glutaraldehyde in pH 2 7.2 buffer). Cords were then removed, further fixed with 1% osmium tetroxide and alcohol gradient, and then coronal sections were taken and placed in resin, stained with Toluidine Blue, and then areas of lesion were cut into thin section for examination by electron microscope. The person evaluating the micrographs did not know which experimental group was the source for the histological samples. Results Effect of T Cell Depletion on Survival: During the first month after injection of virus, 12% of the immunocompetent controls died with signs of polio-like hind-leg paralysis, and another 25% died in the following two months, with additional symptoms of demyelination. In the T-cell depleted group, 80% died within three weeks after virus injection, while all had died by 50 days afterwards (Fig. 1). These depleted mice showed encephalitis symptoms (hunched posture, hind-limb paralysis, ruffled fur and lowered body temperature. None of the saline-injected controls died. Figure 1. Survival curve of mice injected intracranially with TMEV virus. Solid line at top: uninfected controls. Large-dash line: normal, immunocompetent mice. Dotted line: mice with depleted T cells. N = 10 mice per group. Effect of T Cell Depletion on Viral Replication: Assay of viral content from three mice per group that were killed on days 1, 3, and 7 after infection, revealed that the virus was reproducing in both brain and spinal cord during the first month after infection (Figs. 2 and 3). 3 Fig. 2. Amount of virus particles (“plaques”) in brain cells in normal mice (a) and in T-cell depleted mice (b) at various times after infection. Each dot represents viral count from a single mouse. Curve was hand-drawn from the average count at each time point. Fig. 3. As in Figure 2, except these data are from viral counts in spinal cord. The mice that were infected but without T-cell depletion, developed a dramatic decrease in virus on day 28, a time where high levels of antibody to TMEV had developed (Fig. 4). This was most conspicuous for the IgG antibody that is specific for TMEV. None of these mice showed clinical signs by day 28, but at day 42, three mice became paralyzed and high levels of virus were detected in both their brains and spinal cord. In the late stages of infection (days 56 to 77) virus was isolated from the nervous system of six mice. Two of these mice had clinical signs of demyelination. In the T-cell depleted mice, virus levels peaked at day 14, but unlike mice in the undepleted group, the virus was not cleared at day 28. In both groups of mice, the peak virus level coincided with a peak in symptoms. 4 Fig. 4. Antibody levels to TMVE virus in the mice that provided the data from normal (non-depleted mice) shown in Figures 2 and 3.Data expressed as mean + standard deviation of three or more mice tested at each time point. (a) Average level of IgG antibody (solid line) and IgM antibody (dashed line). The negative signs indicate absence of neutralizing anti-TMEV antibody and the + signs indicate the number of animals with such titres greater than 1/100. (b) Antibody levels to mouse myelin at various times after virus injection. Effect of T Cell Depletion on Virus-specific Antibody Production (data not shown): Assay of serum for class-specific antibody against TMEV revealed presence of IgM antibodies specific to TMEV in non-depleted mice by day 14 and peaking at day 28. Neutralizing antibodies were also present by day 28. However, mice with depleted T cells failed to produce detectable serum antibodies against TMEV. Effect of TMEV Infection on Presence of Antibodies to Mouse Myelin (data not shown): Nondepleted mice developed antibodies to myelin by day 28, peaking between days 48-56. In mice with T-cell depletion, antibody production was undetectable. 5 Effect of T Cell Depletion on the Chronic Demyelination Phase of Infection: In a separate experiment, 11 mice were treated with T cell antibody or a mixture of the four monoclonal antibodies on days 65 and 72 after TMEV infection. These mice were then observed twice weekly for signs of demyelination. The T-cell depletion created by T-cell antibody decreased the number of mice that developed severe clinical signs of demyelination (Fig. 5). Fig. 5. Incidence of demyelination symptoms in mice after infection with virus in nondepleted mice (a), mice treated on day 65 and 72 with T-cell antibody (b), or a mixture of anti-T cell antibodies. N = 11 per group. Clinical signs of demyelination were evident at day 155 days after infection in six of eight nondepleted mice, but only 1 of 8 T-cell depleted mice had such signs. By day 212 after infection revealed, 5 of 8 non-depleted mice had severe demyelination symptoms, but all of the T-cell depleted mice were normal. Over the course of the experiments, 10 of 13 non-depleted mice developed severe signs of demyelination lasting more than a week, but such signs occurred in only 4 of 11 in the T-cell depleted group. Interestingly, in a group where all T cells (not just the L3T4 cells) were depleted, 10 of 11 mice developed clinical disease lasting between 1 and 7 days. Infective virus was isolated only from spinal cords of mice that had clinical signs of demyelination at either 119, 214, or 240 sampling days (data not shown in this adaptation). All infected mice had developed autoantibodies to myelin. T-cell depleted groups had lower antibody levels than non6 depleted mice. Depletion of L3T4 after antibody production has occurred does not affect the antibody response that occurred when B cells were present. The histological examination of 3 to 5 mice per group on days 211 or 240 post infection revealed clear correlation between amount of detectable virus in spinal cord and the presence of clinical signs of demyelination (data not shown here). Discussion The strain of mice used (CBA) was selected because their demyelination process is the most similar to that which occurs in human multiple sclerosis. The spinal cord lesions of TMEV-infected CBA mice have large focal areas of demyelination in white matter areas of lateral and ventral areas of the cord. These areas also show infiltration of immune-response cells such as lymphocytes and macrophages. (references were cited). The experiments reported here revealed four findings of special interest: 1. TMEV infection caused rapid death in this strain of mice if L3T4 T cells were depleted prior to infection. It thus appears that helper T cells help infected mice to recover from TMEV infection. If these T cells are depleted, virus titers rise in cord and brain within one month, whereas in control mice such titers have declined to undetectable levels. The TMEV virus is presumably more potent when T cells are depleted because the animals can no longer get stimulation of B cells to make antibodies. Other workers (references cited) have shown that antibodies do help animals recover from infection with various viruses in this same virus family. 2. In late stages of infection in non-depleted mice, TMEV virus could only be recovered from spinal cords of mice that showed clinical signs of demyelination. This contrasts with the presence of virus in the first month of infection in normal, non-depleted mice. The clearance of virus in late stages of infection in such mice is attributed to IgG antibody which peaks late in the infection. 3. Autoantibodies against myelin appeared in late stages of infection. This may have resulted from viral damage to antibodies or the antibodies may have actually contributed to the myelin damage. Previous cited literature is unclear on this point. However, in this study, there was no clear correlation between antibody titer and amount of myelin damage. Moreover, elevated myelin antibody levels occurred in infected but clinically and histologically normal mice. A strong relationship between virus presence and demyelination was indicated by the observation that virus could be detected in the spinal cord of mice showing demyelination. 4. The incidence of disease was reduced in mice in which L3T4 cells or all T cells were depleted at day 65 or 72. In the late demyelination stage of infection in normal mice, virus could not be isolated from spinal cords of mice without signs of demyelination, but was consistently recov7 ered from mice that showed such symptoms. T-cell depletion reduced both clinical and histological signs of demyelination. Prior research (cited) on the mechanism by which TMEV causes demyelination has not provided a clear explanation. Some have suggested that the virus kills the glial cell type (oligodendrocytes) that create myelin. This is consistent with the present observation that virus is present in demyelinated regions of spinal cord at the time when clinical symptoms are evident. Another theory is that virus may be “re-activated” when T cells invade infected nervous system. The present study’s demonstration of reduced demyelination symptoms when T cells were depleted supports another lab’s report that antibodies to thymus gland (where T cells are made) reduce TMEV-induced demyelination. The unresolved issue is whether the T cell effect is autoimmune. The authors concluded that T cells play a double role in TMEV infections. They are necessary to recovery from an acute infection, presumably because they help B cells make anti-viral antibody. However, T cells may promote the delayed demyelination that occurs in chronic infection. Glossary Antibody: a molecule, usually protein, that is produced by cells of the immune system in response to exposure to a “foreign” antigen, that is one not usually found in that individual that develops the immune response. Antigen: a molecule, usually protein, that evokes an immune response. Autoimmune disease: a disease in which an immune response develops to self-antigens. It is like being allergic to yourself. B cells: a specific type of lymphocyte that produces antibodies. Demyelination: a disease state in which there is destruction of the glial cells that make up myelin. Function of the adjacent neurons is disturbed by demyelination. ELISA test: a biochemistry assay that uses antibodies and color change to identify presence of antigen. Encephalomeylitis: an inflammatory disease that affects both neurons and their supporting glial cells. Glial cells: the most common type of cell in the nervous system that vastly outnumbers neurons. They support neuron function, and one cell type, oligodendrocytes, wrap their membranes around neurons to provide a degree of electrical insulation. This wrapping is called “myelin.” Immune response: production of T cells and antibodies in response to the presence of antigen molecules. Monoclonal antibody: a homogeneous batch of antibodies in which all the proteins are homogeneous because they are made from identical immune cells that are clones of a unique parent cell. This production process increases the specificity with which the antibodies bind to antigen. 8 Sucrose Gradient Separation: a differential centrifugation procedure in which a sucrose gradient is created by gently overlaying lower concentrations of sucrose on top of higher concentrations in a centrifuge tub. A homogenized tissue is applied at the top of the gradient, and high-speed centrifugation separates out components of the sample in various layers of the gradient. For virus isolation, the virus can be largely separated and purified from other cell constituents and will concentrate in one specific area of the gradient. T cells: a specific type of cell (lymphocyte) in the immune system, found mostly in the thymus, a gland in the upper-chest, lower neck region. “Helper T cells” are those that stimulate (help) so-called B cells to produce antibodies. They do this indirectly by the release of small molecules known as cytokines. White-matter areas: Regions of spinal cord (and brain) where there are relatively large amounts of myelin. In unstained histological sections, these areas appear relatively white, whereas neurons or their processes appear grey. 9