Resident macrophages (ramified microglia) of the adult brown Norway rat central nervous system are constitutively major histocompatibility complex class II positive.

A flow cytometric phenotype for isolated adult central nervous system (CNS) ramified microglia was previously defined (CD45low CD11b/c+) in the Lewis strain rat, that clearly distinguished these cells from all blood-derived leucocytes, the latter being CD45high. Consistent with the reported lack of major histocompatibility complex (MHC) expression in the CNS, isolated microglia were mostly MHC class II-. Employing these phenotypic criteria, we now show that a proportion of microglia in Brown Norway (BN) strain rats are constitutively MHC class II+. In spinal cord, up to 25% of microglia are distinctly positive and most have some level of expression. In situ staining of MHC class II+ microglial cells in BN rats indicates that positive cells are typical of ramified microglia on the grounds of both morphological appearance and anatomical location. In Lewis (LEW) rats, the few MHC class II-expressing cells isolated from the normal CNS are CD45high blood-derived cells and not resident microglia. After infection of both LEW and BN rats with a neurotropic murine hepatitis virus (MHV-JHM), MHC class II was rapidly upregulated on microglia in the BN but not in the LEW strain. In the latter, inflammatory cells were the predominant MHC class II-expressing population. Nevertheless, most microglia in the LEW strain could, after some delay, be induced to express MHC class II after transfer of an experimental autoimmune encephalomyelitis (EAE)-inducing encephalitogenic T cell line. Paradoxically, strains resistant to EAE (exemplified by the BN) contained more constitutive MHC class II-expressing microglia than susceptible ones, when a variety of strains were examined. The results clearly establish that the normal CNS may contain MHC class II-expressing cells that are a resident rather than a transient blood-derived population. It is significant that this expression is strain related, but there is no evidence that microglial cell constitutive MHC class II expression predisposes to EAE susceptibility.

The role of T-cell-mediated mechanisms in virus infections of the nervous system.

T lymphocytes play a decisive role in the course and clinical outcome of viral CNS infection. Summarizing the information presented in this review, the following sequence of events might occur during acute virus infection: After invasion of the host and a few initial rounds of replication, the virus reaches the CNS in most cases by hematogeneous spread. After passage through the BBB, CNS cells are infected and replication of virus in brain cells causes activation of the surrounding microglia population. Moreover, local production of IFN-alpha/beta induces expression of MHC antigens on CNS cells, and microglial cells start to phagocytose cellular debris, which accumulates as a result of virus-induced cytopathogenic effects. Upon phagocytosis, microglia becomes more activated; they up-regulate MHC molecules, acquire antigen presentation capabilities and secrete chemokines. This will initiate up-regulation of adhesion molecules on adjacent endothelial cells of the BBB. Transmigration of activated T lymphocytes through the BBB is followed by interaction with APC, presenting the appropriate peptides in the context of MHC antigens. It appears that CD8+ T lymphocytes are amongst the first mononuclear cells to arrive at the infected tissue. Without a doubt, their induction and attraction is deeply influenced by natural killer cells, which, after virus infection, secrete IFN-gamma, a cytokine that stimulates CD8+ T cells and diverts the immune response to a TH1-type CD4+ T cell-dominated response. Following the CD8+ T lymphocytes, tissue-penetrating, TH1 CD4+ T cells contact local APC. This results in a tremendous up-regulation of MHC molecules and secretion of more chemotactic and toxic substances. Consequently an increasing number of inflammatory cells, including macrophages/microglia and finally antibody-secreting plasma cells, are attracted to the site of virus infection. All trapped cells are mainly terminally differentiated cells that are going to enter apoptosis during or shortly after exerting their effector functions. The clinical consequences and the influence of the effector phase on the further course of the infection depends on the balance and fine-tuning of the contributing lymphoid cell populations. Generally, any delay in the recruitment of effector lymphocytes to the tissue or an unbalanced combination of lymphocyte subsets allows the virus to spread in the CNS, which in turn will cause severe immune-mediated tissue effects as well as disease. If either too late or partially deficient, the immune system response may contribute to a lethal outcome or cause autosensitization to brain-specific antigens by epitope spreading to the antigen-presenting system in peripheral lymphoid tissue. This could form the basis for subsequent booster reactions of autosensitized CD4+ T cells--a process that finally will end in an inflammatory autoimmune reaction, which in humans we call multiple sclerosis. In contrast, a rapid and specific local response in the brain tissue will result in efficient limitation of viral spread and thereby a subclinical immune system-mediated termination of the infection. After clearance of virus-infected cells, downsizing of the local response probably occurs via self-elimination of the contributing T cell populations and/or by so far unidentified signal pathways. However, much of this is highly speculative, and more data have to be collected to make decisive conclusions regarding this matter. Several strategies have been developed by viruses to escape T cell-mediated eradication, including interference with the MHC class I presentation pathway of the host cell or "hiding" in cells which lack MHC class I expression. This may result in life-long persistence of the virus in the brain, a state which probably is actively controlled by T lymphocytes. Under severe immunosuppression, however, reactivation of viral replication can occur, which is a lethal threat to the host.

Detection and identification of virus-specific, oligoclonal IgG in unconcentrated cerebrospinal fluid by immunoblot technique.

A technique is described which allows the detection of virus-specific oligoclonal IgG in unconcentrated cerebrospinal fluid (CSF) from patients with virus infections of the central nervous system. CSF is isoelectrically focused in agarose gels and immunoglobulins are blotted to nitrocellulose filters, passively loaded with either anti-human IgG or viral antigen. Transferred total IgG, as well as virus-specific IgG, is identified by the use of peroxidase-labelled anti-human IgG and 4-chloro-1-naphthol as a precipitating peroxidase substrate. Application of this assay in cases of SSPE, mumps meningitis and herpes simplex encephalitis demonstrates sensitivity and possible suitability of this technique for use in diagnosis of virus infections of the CNS.

Comparative analysis of virus-specific antibodies and immunoglobulins in serum and cerebrospinal fluid of subacute measles virus-induced encephalomyelitis (SAME) in rats and subacute sclerosing panencephalitis (SSPE).

The intrathecal humoral immune response was analysed in patients with subacute sclerosing panencephalitis (SSPE) and Lewis rats with subacute measles virus (MV)-induced encephalomyelitis (SAME). SSPE patients as well as SAME rats revealed oligoclonal, intrathecal antibody synthesis with MV specificity. SAME rats synthesized MV-specific antibodies intracerebrally to a higher extent than SSPE patients. Although a restricted isoelectric pattern of MV-specific antibodies was detected in the cerebrospinal fluid (CSF) of SSPE patients as well as of SAME rats, the heterogeneity within clusters of immunoglobulin bands was higher in the rat specimens. Increase in the blood-brain barrier permeability for albumin was exclusively detected in SAME rats but not in SSPE patients. These data suggest that the rat model offers excellent opportunities to study the initial humoral events in MV-induced encephalitides.

Cervical lymphoid tissue but not the central nervous system supports proliferation of virus-specific T lymphocytes during coronavirus-induced encephalitis in rats.

The CD4+ T lymphocyte response in the central nervous system (CNS) and cervical lymph nodes (CLNs) of rats with different susceptibility to coronavirus-induced encephalitis was investigated. The majority of CD4+ T lymphocytes entering the virus-infected CNS in the course of the infection are primed cells that neither proliferate ex vivo nor can be stimulated to proliferation by viral antigens or mitogen in vitro. In contrast, T lymphocytes taken from CLNs of the same animals revealed a strong proliferative response. Restimulation of CLN lymphocytes by viral antigens disclosed a striking difference between the disease-resistant rat strain Brown Norway (BN) and the susceptible Lewis (LEW) strain. Whereas BN lymphocytes responded as early as 5 days post infection, it took more than 11 days until a comparable proliferation was detectable in LEW lymphocytes. From these data we postulate that the majority of T lymphocytes entering the virus-infected brain after sensitisation and expansion in cervical lymph nodes is unresponsive to further proliferation signals and that the kinetics and magnitude of T lymphocyte stimulation in CLNs play an important role in the clinical course of the infection.

Analysis of the intrathecal humoral immune response in Brown Norway (BN) rats, infected with the murine coronavirus JHM.

Serum and CSF specimens from clinically healthy Brown Norway (BN) rats inoculated intracerebrally with corona virus JHM were analysed with respect to the state of the blood-brain barrier (BBB) and the intrathecal synthesis and isoelectric distribution of immunoglobulins (Ig). Increased CSF/serum ratios for Ig in the context of an intact BBB were never seen in the absence of intrathecal synthesis of virus-specific antibodies. Affinity-mediated immunoblot analysis revealed a broad pattern of virus-specific antibodies with embedded clusters of restricted heterogeneity, but no signs of oligoclonal Ig production carrying non-viral specificity. From these data it was concluded that BN rats do control the intracerebral spread of JHM virus effectively by a strong local virus-specific antibody response, thereby preventing a clinically apparent disease.

CD1 expression is differentially regulated by microglia, macrophages and T cells in the central nervous system upon inflammation and demyelination.

Expression of CD1 by microglia, macrophages and T cells was investigated ex vivo. In the healthy central nervous system (CNS), resident microglia, macrophages and T cells express levels of CD1 significantly lower than that expressed by splenic macrophages and T cells. During experimental autoimmune encephalomyelitis (EAE), CD1 expression by microglia and the number of CD1+ microglia increase. Macrophages and T cells strongly upregulate CD1 expression in the CNS, but not in the spleen. Whereas the function of CD1 expressed by T cells remains unclear, the expression by microglia and macrophages provides the CNS with a (glyco)lipidic-presenting molecule in an inflammatory and demyelinating environment.

Murine coronavirus-induced encephalomyelitis in rats: analysis of immunoglobulins and virus-specific antibodies in serum and cerebrospinal fluid.

The humoral intrathecal immune response in coronavirus-induced demyelinating encephalomyelitis in rats associated with an autoimmune reaction to brain antigen, was analysed. The CSF of these animals revealed immune reactions which were directed against coronavirus and other, unknown, antigens. In general, no direct correlation between the disease, the state of the blood-brain barrier (BBB), intrathecal synthesis of Ig and the presence of virus-specific antibodies was detectable, suggesting that the humoral, in contrast to the cellular, immune response does not play a significant pathogenetic role in this CNS disease.

Human immunodeficiency virus infection: affinity-mediated immunoblot detects intrathecal synthesis of oligoclonal IgG specific for individual viral proteins.

An affinity-mediated immunoblot (AMI) is introduced, able to identify virus-specific, oligoclonal IgG in unconcentrated cerebrospinal fluid (CSF) of patients infected by HIV. CSF specimens are isoelectrically focused in agarose gels and blotted to nitrocellulose (NC) strips coated with E. coli recombinant HIV-specific proteins. Virus-specific antibody clones transferred to the filter are detected by solid-phase enzyme immunoassay (EIA). Only 0.5 micrograms IgG is sufficient to demonstrate the clonal distribution of antibodies specific for an individual HIV protein. Intrathecal synthesis of oligoclonal antibodies is seen by AMI, comparing equal quantities of serum- and CSF-derived IgG. Because of its excellent sensitivity and specificity, AMI detects autochthonous HIV-specific IgG synthesis even when comparative EIA fails. Because multiple specimens can be processed within a few hours by AMI, this technique is recommendable for routine diagnosis.

Persistent intrathecal secretion of oligoclonal, Borrelia burgdorferi-specific IgG in chronic meningoradiculomyelitis.

In the cerebrospinal fluid IgG of five patients with lymphomeningoradiculitis (Bannwarth's syndrome) and radiculomyelitis studied by immunoblot technique an oligoclonal pattern was found. Most of these oligoclonal bands were specific for Borrelia burgdorferi. In patients suffering from chronic meningoradiculomyelitis, repeated CSF examination by this technique showed persistent secretion of identical IgG bands. Thus, the specific humoral immune response and the disease activity could be documented over the course of the disease.

Immunoglobulin G subclass preference of intrathecally produced HIV-specific oligoclonal antibodies.

Cerebrospinal fluid (CSF) and serum samples from 16 HIV-seropositive subjects were examined by ELISA and isoelectric focusing for the presence of HIV-specific immunoglobulin G (IgG) subclass antibodies. Autochthonous synthesis of IgG in the CSF was demonstrated in 11 patients. Intrathecally synthesized antibodies specific for individual HIV structural proteins were largely restricted to IgG1 (10 of 11 patients). One patient revealed additional local synthesis of IgG2 antibodies related to HIV-p15. In another patient locally produced antibodies specific for HIV consisted entirely of IgG4. In this patient amounts of locally produced antibodies were too low to be detected by routine procedures (calculation from an evaluation graph). No local synthesis of HIV-related IgG3 was demonstrated in any patient. Further studies will be needed to show whether determination of IgG-subclass-restricted antibodies to HIV is of prognostic value.

Analysis of oligoclonal antibody bands against individual HIV structural proteins in the CSF of patients infected with HIV.

Intrathecal antibody responses to HIV were investigated by a highly sensitive immunoblot assay. Serum and CSF specimens were tested for reactivity with the recombinant HIV gag proteins p15, p17 and p24 and with the recombinant transmembrane protein p41. Autochthonous production of virus-specific IgG to one or more HIV structural proteins was seen in 8 of 10 asymptomatic seropositive subjects, in 3 of 4 men with AIDS-related complex, and in 9 of 13 patients with AIDS. These results were consonant with an elevated CSF/serum antibody ratio to total HIV antigen. The high frequency of local HIV-specific antibody synthesis in seropositive individuals without related clinical symptoms indicates an early involvement of the CNS in HIV infections.

Intrathecal synthesis of virus-specific oligoclonal antibodies in patients with enterovirus infection of the central nervous system.

Cerebrospinal fluid (CSF) and serum samples from six patients with enterovirus infections were investigated by isoelectric focusing (IEF) and affinity-mediated immunoblot AMI) for the clonal distribution of entervirus-specific antibodies. In two patients with either acute meningitis or encephalitis and in one patient with a relapse of multiple sclerosis, oligoclonal IgG bands specific for enteroviruses were found predominantly in the CSF, revealing intrathecal synthesis of these antibodies. In three other patients with neurological symptoms probably unrelated to a current enterovirus infection, IEF and AMI disclosed nearly identical patterns of coxsackievirus-B-specific oligoclonal bands in the CSF and serum, indicating diffusion of these antibodies from the serum into the CSF. Although the number of patients in this study is small, the results suggest that intrathecally synthesized enterovirus-specific antibodies may be used as a means of identifying an enterovirus infection of the CNS.

The effect of transforming growth factor-beta 2-specific phosphorothioate-anti-sense oligodeoxynucleotides in reversing cellular immunosuppression in malignant glioma.

This in vitro study was aimed at restitution of transforming growth factor (TGF)-beta 2-mediated suppression of T-lymphocyte activation within malignant gliomas. In early-passage tumor cell cultures of two glioblastomas (HTZ-153 and HTZ-209) and one malignant astrocytoma classified as World Health Organization Grade III (HTZ-243), autologous peripheral blood mononuclear cells were activated by interleukin-1 alpha and interleukin-2 in vitro (lymphokine-activated killer cells) and tested for cytotoxic and proliferative activity. In expression studies (Western blot and Northern hybridization) of all three tumors, TGF-beta could be detected at the protein and messenger ribonucleic acid (mRNA) levels. A polyclonal anti-TGF-beta neutralizing antibody did not enhance lymphocyte proliferation upon stimulation with tumor targets (3H-thymidine incorporation) and slightly stimulated lymphocyte cytotoxicity against autologous target cells. Preincubation of target cells for 12 hours with TGF-beta 2-specific phosphorothioate-anti-sense oligodeoxynucleotides (S-ODN's) did, however, enhance lymphocyte proliferation up to 2.5-fold and autologous tumor cytotoxicity up to 60%, compared to controls not treated with S-ODN's. Incubation of tumor cells with TGF-beta 2-specific S-ODN's resulted in decreased TGF-beta-specific immunoreactivity in cultured glioma cells, in reduced TGF-beta 2 protein concentration (Western blot), and in a change in the expression pattern of TGF-beta 2 mRNA's. These observations may have implications for in vivo and in vitro activation of a cellular immune response against autologous malignant glioma cells.

Peripheral and intracerebral T cell immune response in cats naturally infected with Borna disease virus.

Borna disease virus (BDV) is a neurotropic agent with capacity to cause encephalomyelitis in a wide range of animal species, including horses and cats. Recent studies also point to a link between BDV and human neuropsychiatric disorders. The pathogenesis of Borna disease (BD) has been proposed to be immune-mediated, mainly through the effects of cytotoxic T cells. We used flow cytometric analysis in order to characterize the peripheral and intracerebral T cell immune response in cats naturally infected with BDV. Our results show the presence of two different CD8+ cell populations (CD8+low and CD8+high) in the blood, spleen and brain of these cats. In the brain, CD8+low cells predominated over CD8+high cells. Since CD8+low cells have been suggested to represent a non-MHC-restricted T cell population, the recruitment of such cells to the brains of BDV-infected cats could possibly be of importance for the clearance of virus from neurones.

Coronavirus JHM induced demyelinating disease: specific domains on the E2-protein are associated with neurovirulence.

Infections of rodents by murine coronaviruses can lead to chronic diseases of the central nervous system. These infections are interesting systems to study mechanisms which could be relevant for the pathogenesis of certain human diseases. One major factor influencing the outcome of infection is related to the virus. To understand the virological basis for neurovirulence we compared JHM-virus isolates with different biological properties. JHM-Wt causes only acute disease, JHM-Ts43 a demyelinating encephalomyelitis and a virus shedded from persistently infected cells (JHM-Pi) is not virulent at all. The spread of these viruses in glial cell cultures reflects their different neurovirulence for animals. The peplomer E2 of these viruses reveals structural and antigenic differences. We characterised the epitopes of E2 with a panel of monoclonal antibodies. Four epitopes are associated with regions important for neutralisation, cell fusion and attachment. More than five epitopes are not related to such functions. Epitopes differ in their location and accessibility on the E2 protein subunits between JHM-Wt, JHM-Ts43 and JHM-Pi. To identify epitopes in regions important for pathogenesis, we performed animal studies with variants selected by monoclonal antibodies. Variants changed in a defined epitope (E2-Ba) induce in Balb/c mice a chronic disease. Variants changed in only one of the other three neutralisation epitopes induce acute disease. These results support and extend the observation that the peplomer protein E2 is a major determinant for virulence and antigenic variability of coronaviruses 1,4,5,6,8,10,17,19,22,23. Increasing evidence had been obtained that certain structural features of this protein are important for the cell tropism of the virus. Furthermore, this protein influences strongly the type and specificity of immune responses against viral and host antigens. The highly advanced knowledge on structure and replication of coronaviruses will be of great value to analyze further mechanisms leading to inflammatory demyelinating diseases associated with a persistent virus infection.