Strategies for the modulation of neuroimmunological disease at the level of autoreactive T-lymphocytes

Cell lines of autoimmune T-lymphocytes have been established in several neuroimmunological model diseases and also in a human neurological autoimmune disease, myasthenia gravis. These cell lines generally have the T helper/inducer phenotype and recognize autoantigen in the context of class II histocompatibility antigens. Autoreactive helper T cell lines may become useful tools for the evaluation of new immunotherapeutic strategies. (1) Treatment with anti-Ia monoclonal antibodies presumably interferes with the interaction between Ia on the surface of antigen-presenting cells and the autoreactive T cell receptor; (2) Therapy with unmodified or modified autoantigen may be used to tolerize or delete the autoimmune T cells; (3) Monoclonal antibodies against the 'T cell domains' of autoantigen may prevent its recognition by the autoreactive T cells; (4) Treatment with monoclonal antibodies against T cell clonotypic or differentiation antigens may effectively delete or inactivate the autoreactive T cells. Furthermore, autoreactive helper T cells may be used to induce and establish anti-idiotypic suppressor T cell lines, or the autoimmune helper T cells may themselves display suppressive effects in an allogeneic system.     

Major histocompatibility antigens and lymphocyte subsets during experimental allergic neuritis in the Lewis rat

Summary Major histocompatibility antigens were identified in frozen sections of normal Lewis rat peripheral nerve tissue with monoclonal antibodies and an avidin-biotin-peroxidase complex system. Class I antigen is normally required for cytotoxic/suppressor T lymphocyte function and class II antigen for activation of helper T lymphocytes. In the sciatic nerves class I antigen was expressed diffusely by most endoneurial and perineurial cells but class II antigen only by a minority. In the cauda equina class I antigen was expressed by all arachnoid and some endoneurial cells, while class II antigen was expressed by a smaller proportion of arachnoid cells in the endoneurium of spinal roots and interstitial cells surrounding dorsal root ganglion neurons. The endothelium of endoneurial, perineurial and meningeal vessels uniformly expressed class I but not class II antigen. Experimental allergic neuritis was induced in Lewis rats by immunisation with bovine intradural root myelin. Early lesions consisted of multifocal infiltration of the nerve roots by cells expressing leucocyte common antigen. Surrounding endoneurial cells showed markedly increased expression of major histocompatibility antigens. In inflammatory lesions about 10% of the cells were stained with pan T cell antibodies. T lymphocyte subsets were identified with antibody W3/25 for helper cells and MRC OX-8 for cytotoxic/suppressor cells. The W3/25 positive cells were usually slightly in excess of OX-8 positive cells and their relative proportions did not alter during the disease. The presence of class I antigen on normal endothelium and its increased expression on endoneurial cells in the early phase of inflammation suggest an important role for class I restricted lymphocytes in the pathogenesis of the early stages of experimental allergic neuritis.     

Progressive expression of immunomolecules on microglial cells in rat dorsal hippocampus following transient forebrain ischemia

Summary We show a differential up-regulation of immunomolecules in the rat dorsal hippocampus accompanying neuronal cell death as a consequence of transient forebrain ischemia (four-vessel occlusion model). Using a panel of monoclonal antibodies (mAbs), we have examined the time course of expression of major histocompatibility complex (MHC) antigens class I (OX-18) and class II (OX-6), leukocyte common antigen (OX-1), CD4 (W3/25) and CD8 (OX-8) antigens, CR3 complement receptor (OX-42), as well as brain macrophage antigen (ED2). The study was performed at time intervals ranging from 1 to 28 days after reperfusion. Throughout all post-ischemic time periods, strongly enhanced immunoreactivity on microglial cells in the CA1 region and dentate hilus and, to a lesser extent, in CA3 was demonstrated with mAb OX-42. MHC class I-positive cells (OX-18) appeared on day 2, whereas cells immunoreactive with OX-1 and W3/25 became evident in the CA1 and hilar regions on post-ischemic day 6. In contrast, MHC class II (Ia) antigen was first detected on indigenous microglia by day 13. In some animals, the OX-8 antibody resulted in the labelling of scattered CD8-positive lymphocytes, but perivascular inflammatory infiltrates were absent. No changes in the expression of ED2 immunoreactivity on perivascular cells could be observed. The results show that following ischemic injury, microglial cells demonstrate a timedependent up-regulation and de novo expression of certain immunomolecules, indicative of their immunocompetence. The findings are compared with those obtained in other models of brain injury.Supported in part by NIH/NINCDS PO 1 NS27511     

Transforming growth factors beta 1 and beta 2: cytokines with identical immunosuppressive effects and a potential role in the regulation of autoimmune T cell function

The transforming growth factors TGF-beta 1 and beta 2 are cytokines with pronounced effects on leukocyte growth and function. To evaluate a potential use of these factors as immunosuppressive agents, we compared the effects of TGF-beta 1 and beta 2 on autoimmune T cells in rat inflammatory central nervous system disease, experimental allergic encephalomyelitis (EAE). We observed that both factors strongly inhibited in vitro activation of autoimmune T cells, suppressed the accumulation of interleukin-2 mRNA and decreased the expression of rat T cell activation antigens. In addition, cyclical changes in susceptibility to TGF-beta was observed with T line cells. The modulation of in vitro T cell function is associated with a considerable suppression of encephalitogenic capacity of autoimmune T line cells. Thus, TGF-beta 1 and beta 2 might have physiologic importance in limiting local T lymphocyte proliferation and effector function in autoimmune disease.     

Expression of immunomolecules on microglial cells following neonatal sciatic nerve axotomy

We have examined the microglial reaction accompanying motor neuron death following sciatic nerve crush in the newborn rat using lectin staining with the Griffonia simplicifolia B4-isolectin, as well as immunocytochemistry with a panel of monoclonal antibodies directed against brain macrophage antigen (ED2), and various immunologically important surface molecules (immunomolecules), such as major histocompatibility complex (MHC) class II (Ia) antigen (OX-6), CR3 complement receptor (OX-42), CD4 antigen (W3/25), and leukocyte common antigen (OX-1). The lectin histochemical method provided the earliest indication of a microglial response by demonstrating increased microglial density and clustering around dying motoneurons as early as 2 days after lesioning. Most immunomolecules were largely undetectable in the normal and early post-lesion spinal cord; however, at post-lesion day 5 localized expression of Ia antigen was visualized in the area of degenerating motor neurons. Ia expression preceded the appearance of other immunomolecules at day 8. No increase in staining with the ED2 antibody for macrophage antigen could be detected at any post-lesion interval. When compared to the microglial activation that occurs after axotomy in adult animals, our results show a similar onset in microglial activation in neonatal animals; however, the duration of immunomolecule expression is much briefer.     

Expression and distribution of various antigens of developing microglial cells in the rat telencephalon.

The distribution of microglia during the early stages of postnatal development in the rat was studied on rat brain from day of birth to postnatal day 90 (P90), using immunohistochemical methods with a panel of monoclonal antibodies that recognized the complement type 3 receptor (OX-42), macrophage antigen of unknown function (ED1), and the major histocompatibility complex (MHC) class I (OX-18) or class II (OX-6) antigens. Starting from the day of birth, ameboid microglia can be differentiated with positive immunoreactivity to OX-42, OX-18, and ED1. Labeled cells were localized mainly in the developing white matter. After P21, only positive reaction to OX-42 was present, and those cells had the typical morphology of the resting microglial cells that were located either in the white or grey matter. The changes in the appearance of different antigens are correlated with the morphological differentiation and transformation of ameboid microglial cells that are to become ramified microglia, present in the adult animals.     

Adoptive transfer of experimental allergic encephalomyelitis with lectin-activated spleen cells. Part 2. Studies on T cell subsets and interleukin 2 production

Experimental allergic encephalomyelitis (EAE) can be transferred by spleen cells stimulated in vitro with D-mannose-binding lectins but not with N-acetyl-D-galactosamine (GalNAc)-binding lectins. EAE could also be passively transferred by spleen cells following incubation with wheat germ agglutinin (WGA), in which case the disease transfer was abolished by the specific hapten inhibitor, N-acetyl-D-glucosamine. In the presence of rat T cell monoclonal antibody, either W3/25 or OX-8, both concanavalin A and Wisteria floribunda agglutinin stimulated helper and suppressor T subpopulations. On the other hand, GalNAc-binding lectins were less effective than D-mannose-binding lectins in generating interleukin 2 (IL2) in the culture supernatant, whereas WGA-stimulated spleen cells did not produce IL2. Furthermore, spleen cells cultured with pure IL2 could not transfer EAE to the recipients. These data suggest that some factors distinct from IL2 are required for the differentiation of EAE-effector precursors into the final effector cells in this transfer system.     

Monoclonal antibodies reveal sagittal banding in the rodent cerebellar cortex

We have produced two monoclonal antibodies against polypeptide-associated antigens of developing rat cerebellum. One antibody recognizes an antigen associated with synaptic vesicles and another binds to a polypeptide which is restricted to the cytoplasm of a subset of cerebellar Purkinje cells. Both antibodies reveal the biochemical differentiation of the rodent cerebellar cortex into antigenically distinct sagittal zones.     

Monoclonal antibodies selective for the functional states of bovine factor V and factor Va

Hybridoma technology has been used for the production of murine monoclonal antibodies to bovine coagulation Factor V and its thrombin-activated product, Factor Va. Hybrid cell cultures were assayed for the production of anti-Factor V and anti-Factor Va antibodies by a solid-phase radioimmunoassay. Antibody-producing cell lines were selected, cloned and grown as ascites tumors. Gel filtration chromatography (Ultrogel AcA34) and affinity chromatography (protein A-Sepharose) were used to isolate the monoclonal immunoglobulins from the ascites fluids. Thirteen monoclonal antibodies have been characterized with respect to their binding to Factor V and Factor Va and their effect on cofactor bioactivity. Six of these thirteen antibodies react with both Factor V and Factor Va. One of these antibodies is strongly inhibitory, while a second antibody is only moderately inhibitory. The antibody produced by another cell line binds Factor V but not Factor Va and is not inhibitory. The remaining six cell lines each produce an antibody that reacts preferentially with Factor Va, and each of these antibodies is inhibitory to some extent. Both a radioimmunoassay and light scattering have been used to study the interaction of the immunoglobulins with Factor V and Factor Va. The light scattering technique has proven useful to study the interaction of isolated antibodies and antigens and permits the determination of interaction stoichiometries. Each of the interactions studied was characterized by a stoichiometry of two antigens per antibody. These monospecific immunochemical reagents will be useful in the study of structure and function relationships of Factor V, Factor Va and activation fragments.     

Epitopes common to trypanosomes (T. cruzi, T. dionisii and T. vespertilionis (Schizotrypanum]: astrocytes and neurons

An autoimmune mechanism is commonly invoked to explain the occurrence of neuronal destruction in the chronic phase of Chagas' disease. Monoclonal antibodies raised against T. dionisii (DION) and T. vespertilionis (VESP), and cross-reactive with T. cruzi recognize antigens in cultured cerebellar cells from embryonic and postnatal mice, as revealed by indirect immunofluorescence. Astrocytes (labelled with rabbit anti-GFAP antibody) showed positive reactions with DION 12.7, VESP 8.2 and VESP 9.3 while neurons (labelled with either tetanus toxin or anti-neuron-specific enolase antibody) reacted with the monoclonal antibody DION 10.1b. VESP 6.2 reacted with living cells of a subpopulation of neuronal or unidentifiable cell type. These cross-reactions may explain why not only neurons but also astrocytes may be involved in the autoimmune damage.     

Immortalization and characterization of rat microglial cells

Microglial cell lines from rat brain were established by transfer of a temperature sensitive simian virus 40 large tumour antigen by means of a retrovirus. Four weeks after infection, colonies were generated in the presence of neomycin and granulocyte-macrophage colony stimulating factor (GM-CSF), and subsequently subcloned. Both bulk cell lines and clones proIiferate actively at 33°C. whereas the rate of division was significantly decreased at 39°C when the large T antigen is non-functional. At 39°C these cells take on the microglial phenotype as demonstrated by immunoreactivity to ED-1 (an intra-cellular antigen), OX-42 (complement type 3 receptor), W3/25 (CD4 homologue), OX-6 (MHC class II antigen) and OX-18 (MHC class I antigen). These cells are capable of active phagocytosis and retain these properties for 10–15 passages. Long-term culture of these lines and clones, greater than 15 passages, displayed a gradual down-regulation of all cell surface specific antigens that were not rescued by lipopolysaccharide (LPS), interferon-gamma (γ-IFN), GM-CSF or colony-stimulating factor-1 (CSF-1). The expression of the SV-40 large T antigen was unaffected. These results demonstrate the feasibility of immortalizing short-term cell lines with the SV-40 large T antigen for their use in the characterization of microglial properties.     

Murine monoclonal anti-myelin basic protein (MBP) antibodies inhibit proliferation and cytotoxicity of MBP-specific human T cell clones

Myelin basic protein (MBP)-specific T cell clones, isolated from two patients with multiple sclerosis, expressed the CD4+ phenotype and induced MBP-dependent cytolysis of autologous Epstein-Barr virus (EBV)-transformed B cells. The proliferation and cytolytic activity of the T cell clones were inhibited by four of a panel of five murine monoclonal anti-MBP antibodies in a dose-dependent manner. An isotype-matched antibody with an irrelevant specificity did not have such an effect. These MBP-specific monoclonal antibodies did not block phytohemagglutinin-induced T cell proliferation or allospecific cytotoxicity. These results suggest that some antibodies directed at the autoantigen MBP may play a regulatory role in T cell activation, rather than a pathogenic role, for which there is currently little supporting evidence.     

An in vitro study on the involvement of LINGO-1 and Rho GTPases in Nogo-A regulated differentiation of oligodendrocyte precursor cells

Nogo-A has been considered as one of the most important myelin-associated axonal regeneration inhibitors in the central nervous system. Recent studies have demonstrated various additional physiological roles of Nogo family members. To understand the possible effect of Nogo-A on the differentiation of oligodendrocytes, antibodies against distinct extracellular domains of Nogo-A were applied in cell cultures. Oligodendrocyte precursor cells from P2 rat cortex were grown in the presence of monoclonal antibody against the N-terminal inhibitory domain of Nogo-A or the C-terminal 66 amino acid loop of Nogo-A for 3 days, and the antibody treatment resulted in stunted process extension and inhibited differentiation of oligodendrocytes. Concomitant with morphology changes, Rho GTPases activity was greatly increased upon the antibody treatment and the expression level of LINGO-1, which was recently shown to be a negative regulator for the oligodendrocyte maturation, was upregulated in the process of antibody treatment. These results indicate that endogenous Nogo-A expressed in oligodendrocyte may act though Rho GTPase and LINGO-1 to influence the morphological differentiation of oligodendrocytes and will help us to understand the physiology role of Nogo-A in oligodendrocyte biology.     

T-lymphocytes in experimental autoimmune myasthenia gravis. Isolation of T-helper cell lines

The role of T-lymphocytes in Experimental Autoimmune Myasthenia Gravis (EAMG) was investigated. We generated highly purified, acetylcholine receptor (AChR)-specific T-cell populations and subsequently characterized these cell lines with respect to their membrane phenotype and their function. Using a series of mouse monoclonal antibodies directed against rat lymphocyte surface differentiation antigens, the vast majority of line cells was shown to express a leucocyte common antigen, a T-common antigen and a T-helper antigen. Small subpopulations were Ia or T suppressor antigen-positive. Adaptive transfer to sublethally irradiated, thymectomized recipients revealed that 1 X 10(6) AChR-specific line cells could cooperate effectively with 10 X 10(6) AChR-primed, complement (C3) receptor-bearing (B-cell enriched) spleen cells in the production of anti-AChR autoantibodies. Recipients of B-cells along with relevant line cells developed an acute myasthenic syndrome 6-7 days after cell transfer. Electron-microscopical examination revealed the typical features of "acute phase" EAMG with heavy mononuclear infiltration. There was, however, no evidence antibody-independent cytotoxic activity exerted by AChR-specific line cells.     

Diagnosis of CNS lymphoma using immunofluorescent phenotyping of CSF mononuclear cells

We describe the use of a panel of monoclonal antibodies, directed against leukocyte surface antigens to characterize CSF mononuclear cells with regard to malignancy when cytopathology was inconclusive. Cytocentrifuged preparations from three patients in which traditional modalities had not yielded a diagnosis were studied, utilizing a panel of antibodies for B and T cell antigens. All three patients were found to have B cell lymphoma of the CNS. Rapid institution of the appropriate therapy resulted in marked improvement of CNS symptoms in each case. Our results indicate that in patients with CNS disease and CSF pleocytosis of undefined nature, this technique may provide rapid and precise diagnostic information.     

Down-regulation of complement receptor 3 and major histocompatibility complex I and II antigen-like immunoreactivity accompanies ramification in isolated rat microglia

Isolated primary microglia are highly activated in conventional culture systems. This has restricted studies to the use of late stage measures of activation rather than highly sensitive immunophenotypic and morphological criteria that mark even very early stages of microglial activation in vivo. In the present study, serum-free, serine- and glycine-free medium and poly-L-lysine coated surfaces have been used to demonstrate for the first time isolated rat microglia which (i) downregulate their immunoreactivity for antibodies recognizing complement receptor 3 and major histocompatibility complex antigens while differentiating into ramified cells, and (ii) respond to a subset of modulators with upregulation of complement receptor 3-like immunoreactivity. During 2 weeks of culturing under basal conditions, ramification was accompanied by strong downregulation of OX-42, OX-18 and OX-6 immunoreactivity (antibodies recognizing complement receptor 3 and major histocompatibility complex class I and II antigens, respectively). Ramified cells had lower level immunoreactivity for all three markers than non-ramified cells. High OX-42 immunoreactivity was also associated with morphological signs of activation previously described in vivo. Enhanced OX-42 immunoreactivity was induced by applying either serine and glycine or lipopolysaccharide (LPS) while granulocyte macrophage-colony stimulating factor increased cell number without affecting OX-42 immunoreactivity. LPS induced alterations were apparent within 24 h, were transient, and did not include changes in OX-18 or OX-6 immunoreactivity, cell number or proportion of ramified cells. The results attest to the special efficacy of this culture method for the investigation of the early microglial reaction by use of highly sensitive immunophenotypic criteria.     

Vβ CDR3 motifs associated with BP recognition are enriched in OX-40+ spinal cord T cells of lewis rats with EAE

Spinal cord (SC) T cells were isolated at the onset of actively induced experimental autoimmune encephalomyelitis (EAE) and sorted for the presence of the OX-40 activation marker. Previously, we reported an enhanced bias in Vβ8.2 expression as well as enhanced proliferative responses to basic protein antigens among the OX-40⁺ SC T cells. Here we demonstrate that CDR3 motifs associated with EAE are present at a significantly higher frequency in Vβ8.2 sequences of OX-40⁺ SC T cells (16/17) compared with those of OX-40⁻ SC T cells (5/17). Thus, the OX-40 antigen may be useful as a marker to isolate and characterize autoantigen-specific T cells from the site of inflammation in T-cell-mediated autoimmune diseases. © 1996 Wiley-Liss, Inc.     

Inhibition of PC12 Cell Attachment and Neurite Outgrowth by Detergent Solubilized CNS Myelin Proteins

Adhesion and neurite outgrowth of PC12 cells, as well as the spreading of 3T3 fibroblasts, were inhibited in a dose dependent manner by detergent solubilized mouse central nervous system myelin proteins as a tissue culture substrate. These inhibitory effects could be neutralized by the monoclonal antibody IN-1 directed against the neurite growth inhibiting proteins NI-35 and NI-250. Separation of the detergent soluble proteins of bovine spinal cord by an anion exchange column showed that the peaks of inhibitory activity for the two cell lines overlapped, such that the PC12 cells were inhibited by a larger number of fractions comprising those inhibitory for 3T3 cells. Neurite outgrowth of PC12 cells was not influenced by the myelin associated glycoprotein, MAG.     

Ultrastructural and immunocytochemical studies of macrophages in an excitotoxin induced lesion in the rat brain.

An epidural application of kainic acid (KA) over the cerebral cortex in rat resulted in an extensive lesion in the ipsilateral cerebral cortex. This procedure elicited an accumulation of a large number of macrophages at the site of lesion covering a period of 4 weeks beginning 4 days after the KA application. The macrophages in the centre of lesion were characterized by abundant cytoplasm containing a variable number of lysosomes and phagosomes. Neurons at the same site were depleted during the period examined. They underwent degeneration following the KA treatment. With the monoclonal antibodies OX-42, OX-18 and OX-6, intense immunoreactivity was observed in these cells at the light and electron microscopic levels. Besides these antibodies, the cells were stained positively with the isolectin Griffonia simplicifolia (GSAI-B4). At the periphery of the lesion, many cells bearing the external morphology of microglia were also intensely stained with the GSAI-B4 and the monoclonal antibodies. It was concluded from this study that neuronal degeneration, caused by the excitotoxin KA, induced the accumulation of macrophages which exhibited CR3 receptors (marked by OX-42), MHC I antigen (marked by OX-18) and MHC Ia (marked by OX-6). The expression of these surface antigens may be related to their active phagocytic activity. The reaction with GSAI-B4 indicates the presence of specific lectin receptors on the macrophages which would serve a similar function. The present lectin histochemistry and immunohistochemical studies suggest that macrophages in the centre of the KA-induced lesion were derived from infiltrated monocytes while those at the periphery originated from the activation of local microglial cells.     

T cell line-mediated EAE: prevention and therapy by a monoclonal antibody specific for T lymphoblasts

We studied the effect of a novel monoclonal antibody, pta-3, on T cell line-mediated experimental allergic encephalomyelitis (EAE). The antibody is specific for a differentiation antigen expressed by activated rat T lymphocytes and is cytotoxic in the presence of rat complement for a minor subpopulation of leukocytes, including encephalitogenic T cells (Schluesener et al. 1986). Single intraperitoneal injections of antibody effectively prevented or abrogated lethal EAE. Therapeutic effect was dependent on time of treatment, but even established disease could be cured. Due to the specificity of the antibody for the disease inducing lymphocytes, no side effects of treatment could be observed in living animals or by autopsy.