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Vaccination with naked DNA represents a therapeutic strategy currently under consideration in multiple sclerosis (MS). In this study, we tested the potential therapeutic effect of vaccination with a naked DNA construct encoding proteolipid protein (pRc/CMV-PLP) upon the outcome of subsequent sensitization for experimental autoimmune encephalomyelitis (EAE) actively-induced in SJL mice with PLP139-151 peptide in adjuvant. Intramuscular vaccination with the naked DNA pRc/CMV-PLP construct led to PLP expression in local muscle tissue that persisted for about 8 weeks. Early sensitization for EAE (4 weeks after DNA vaccination) caused recipient mice to develop a severe, exacerbated form of disease (in comparison to control mice), while late sensitization (>10 weeks) resulted in a milder, ameliorated form. In the groups sensitized <10 weeks post-DNA vaccination with pRc/CMV-PLP induction of a Th1-type cytokine response was noted. In contrast, sensitization >10 weeks post-DNA vaccination led to peripheral tolerance as evidenced by a decrease in T cell proliferation and cytotoxic T cell response, no Th2 response, and no increase in apoptosis. These data are novel in that they demonstrate a differential effect of DNA vaccination and have important implications for its use as a mechanism to enhance or modulate immune reactivity.

Human white matter from non-neurologic cases, multiple sclerosis (MS) and other neurologic diseases (OND, inflammatory and non-inflammatory), was subjected to immunocytochemistry and Western blotting for expression of the neuregulin, glial growth factor-2 (GGF2), and its receptors, erbB2, erbB3 and erbB4. GGF2 has previously been shown to have mitogenic effects upon oligodendrocytes in vitro and an enhancing effect upon remyelination in animals with autoimmune demyelination. In all types of human white matter examined, expression of the ligand GGF2 and its three receptors was consistently found on oligodendrocytes, with higher levels being seen in cases of MS. Expression was also seen, albeit at lower levels, on astrocytes and microglial cells, the latter most commonly in MS and OND. In human lymph node tissue, some lymphocytes were positive for erbB2, erbB3 and erbB4. Western blots confirmed the presence of all three receptors in normal, MS and OND white matter. GGF2 and erbB receptor expression did not correlate with areas of remyelination and reactivity occurred throughout the tissue, with some increase in intensity at the edge of MS lesions. Examination of precursor oligodendrocyte immunoreactivity (with anti-PDGF-Ralpha and NG2), revealed widespread expression throughout both normal and diseased white matter. The presence of GGF2 and its receptors on oligodendrocytes and lymphocytes render this cell type a candidate for functional signaling via this pathway, perhaps in relationship to myelinating activity.

We wished to study how infections might trigger relapses of autoimmune diseases such as multiple sclerosis (MS) and encephalomyelitis (EAE). We hypothesized that immune responses to heat shock proteins (hsp) induced by an infection could modulate responses to autoantigens. We induced extra-neuraxial inflammation in SJL mice housed either in specific-pathogen free (SPF) or conventional facilities. Mice in conventional housing are continuously exposed to large numbers of infectious agents. Spleen cell proliferative responses to human HSP60 and bacterial HSP65 were measured as were numbers of cells secreting IFN-gamma or IL-5. Proliferative responses to HSP60 were increased in conventionally housed mice compared to SPF mice and this was associated with skewing of secreted cytokines toward a Th2 pattern. Skewing toward a Th1 pattern was noted in SPF mice. Acute and relapsing EAE was induced in both groups of mice. Acute EAE was, in general, equivalent in all groups. However, SPF mice had more severe relapses than did conventionally housed animals and these differences were amplified by extra-neuraxial inflammation. Immunocytochemical analyses of brains from mice with relapsing EAE showed that increased numbers of brain gamma/delta cells were associated with disease remission. Our data suggest that frequent exposure to infectious agents leads to a relative Th2 skewing of immune responses to hsp and that this is associated with milder, less frequent relapses of EAE. They also support the concept that immune responses to hsp are of potential importance in exacerbating and perpetuating organ-restricted autoimmune diseases.

Proteolipid protein (PLP), a transmembrane protein expressed only in the central nervous system (CNS), is a candidate target autoantigen for autoimmune-mediated demyelination. We have evaluated the effect of a recombinant form of the PLP protein, delta PLP4, in a murine model of experimental autoimmune encephalomyelitis (EAE). PLP-specific T-cell responses were observed following immunization of SJL/J, PL/J and SWR mice with delta PLP4, demonstrating processing of the protein to several distinct antigenic epitopes. Clinical EAE associated with inflammation and demyelination in the CNS also developed after sensitization of mice with delta PLP4 in adjuvant. Conversely, tolerance to delta PLP4 in adult mice and prevention of PLP peptide 139-151-induced EAE was induced by intravenous injection of soluble delta PLP4. The prevention of disease onset was paralleled by a significant reduction in demyelination and CNS inflammatory cell infiltration and diminished PLP139-151-specific T-cell proliferative responses. These results are consistent with the establishment of peripheral T-cell tolerance and reinforce the notion that recombinant myelin antigens and intravenous tolerance induction may prove useful in the modulation of the human demyelinating disease, multiple sclerosis (MS).

It was recently demonstrated that selective phosphodiesterase type 4 (PDE4) inhibition suppresses the clinical manifestations of acute experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), and inhibits the production of tumor necrosis factor-alpha (TNF-alpha), a pathogenetically central cytokine. Since the most common presentation of MS in humans is a relapsing-remitting course, we investigated the therapeutic potential of PDE4 inhibition in the relapsing-remitting EAE model of the SJL mouse. Administration of rolipram, the prototypic PDE4 inhibitor, reduced the clinical signs of EAE during both the initial episode of disease and subsequent relapses. In parallel, there was marked reduction of demyelination and also less inflammation throughout the central nervous system (CNS) of rolipram-treated animals. Gene expression of proinflammatory cytokines in the CNS was reduced in most of the rolipram-treated animals. Additional experiments demonstrated that PDE4 inhibition acted principally by inhibiting the secretion of Th1 cytokines, however, the encephalitogenic potential of myelin basic protein-specific T cells was not impaired. Our findings suggest that PDE4 inhibitors are a promising cytokine-directed therapy in chronic demyelinating disease.

The cytokine lymphotoxin (LT) is known to exist in two forms, secreted LT alpha and a membrane-bound LT alpha/beta complex. LT alpha shares the same receptor as tumor necrosis factor alpha and LT beta is recognized by its receptor, LT betaR. Since LT has been associated with oligodendrocyte pathology, the present study has examined the expression of these molecules by immunocytochemistry in diseased and normal CNS tissue, with a panel of monoclonal antibodies (mAb) to LT alpha, LT beta and LT betaR. Of three mAb to LT beta, two (B27 and C37) gave specific membrane staining on astrocytes, as well as lymphocytes. The third anti-LT beta mAb, B9, was selectively immunoreactive for oligodendrocytes, suggesting specific recognition sites. The reactivity was not specific for multiple sclerosis (MS) since oligodendrocytes in normal and non-MS CNS tissue also displayed positivity. MAb to LT betaR reacted with astrocytes only, giving a punctate membrane staining pattern suggestive of receptor sites. MAb to LT alpha gave strong reactivity on lymphocytes in active MS lesions and weak reactivity on microglia within lesion areas. These results show that mAb to LT alpha and LT beta recognize different cell types within the CNS. Furthermore, individual mAb against LT beta were capable of distinguishing between astrocytes and oligodendrocytes, perhaps indicative of different epitopes on LT beta. The presence of LT betaR on astrocytes suggests possible interactions between infiltrating lymphocytes and astrocytes via the LT pathway.

The mechanisms involved in the elimination of oligodendrocytes and myelin from the demyelinated plaque of multiple sclerosis (MS) are inextricably intertwined and yet most investigations tend to consider them separately. This short review revisits the problem of oligodendrocyte pathology in MS and attempts to put the topic into perspective by examining the numerous immunologically-active molecules associated with the oligodendrocyte, some, but not all, cross-reactive with myelin. The consensus of opinion is that myelin is the primary target in MS but that oligodendrocytes are eventually lost from the lesion. Reappraisal of recent and past works brings into focus a possible key role for soluble mediators, in particular antibody and the pro-inflammatory cytokine, TNF alpha, in oligodendrocyte loss and myelin in MS. Despite extensive neuropathologic investigation by a number of laboratories, no evidence has yet been found to support the concept that apoptosis might account for oligodendrocyte depletion in MS, even though molecules belonging to the apoptotic cascade can be expressed by oligodendrocytes in and around lesions. Indeed, abundant evidence has been presented to show that oligodendrocytes initially respond to the demyelinating insult in MS by proliferating and elaborating new myelin but, no doubt due to the relentless progression of inflammatory events, the cells are eventually lost, probably via a cytolytic pathway. Strategies to block the progression of CNS inflammation in EAE and MS appear to promote the survival of oligodendrocytes and to enhance remyelination. Such strategies appear to hold much promise for the MS patient.

Recent observations have shown that CD1 molecules act as restriction elements in the presentation of antigens to specialized subsets of T cells. To examine the expression of CD1 molecules in multiple sclerosis (MS) lesions, frozen sections of central nervous system (CNS) tissues from nine MS and three other neurological disease (OND) patients, one patient with Wilson's disease, and one non-neurological control were stained by immunocytochemistry. In chronic-active MS lesions, CD1b immunoreactivity was prominent on perivascular inflammatory cells whereas macrophages within the lesion showed little reactivity. At the lesion edge, intense immunoreactivity for CD1b was found on hypertrophic astrocytes. High level expression of CD1b in MS lesions was found to colocalize with the presence of GM-CSF in astrocytes. In chronic-silent lesions, CD1b expression was found on only a few perivascular astrocytic foot processes and the occasional perivascular macrophage. CD1b was not found in the tissues studied for control purposes. In contrast, MHC class II expression was detected on microglia in all tissues examined. The relatively low level expression of CD1b in normal-appearing tissues, chronic-silent lesions and in the OND controls supports the conclusion that the expression of CD1b in active MS lesions is significantly upregulated and could contribute to lesion development.

The discovery of T lymphocytes reactive to the peptide encoded by exon 2 of the myelin basic protein (MBP) gene in multiple sclerosis (MS) patients has drawn attention to MBP isoforms harboring that peptide as candidate autoantigens. Previously, immunological studies in MS had almost exclusively used the more abundant 18.5 kDa isoform of MBP, which does not contain the exon 2 peptide. Investigations of experimental allergic encephalomyelitis (EAE) have also focussed on the 18.5 kDa MBP isoform and its peptides. Since EAE is an animal model widely used to study MS, we examined the encephalitogenic potential of exon 2 peptide in the SJL/J mouse. Evidence for increased expression of exon 2-containing isoforms during remyelination in mouse CNS suggested that exon 2-sensitized T cells, with encephalitogenic capacity, might be important in the perpetuation of relapsing EAE (rEAE). Our experiments have demonstrated that exon 2 peptide is inherently immunogenic in SJL mice and that EAE could be induced by the adoptive transfer of exon 2-sensitized lymphocytes. Furthermore, the disease could be accentuated by the transfer of short-term exon 2-reactive lines or by a combination of adoptive transfer and antigenic challenge with exon 2 peptide. The immunodominant epitope(s) appeared to localize to the segment bordered by amino acids 59-85.

Chronic relapsing experimental allergic encephalomyelitis (EAE) was induced in Thy-1.1 congenic SJL/J mice by the adoptive transfer of myelin basic protein (MBP)-responsive lymph node cells from Thy-1.2 SJL/J mice. The Thy-1 congenic mouse strain was constructed on the SJL (Thy-1.2) background by the initial cross with the AKR (Thy-1.1) strain and does not reject Thy-1.2+ T cells. Quantitative immunocytochemical analysis of the central nervous system (CNS) of Thy-1.1 recipients showed preferential trafficking of Thy-1.2+ T cells to the meninges and white matter, beginning prior to onset of clinical signs. At 7 days post-transfer (dpt), Thy-1.2+ donor cells constituted 2.5% of the infiltrating cells and reached peak values (ca. 10%) during the first attack. At later stages (up to ten relapses), Thy-1.2+ T cells constituted 2-5% of the infiltrate. In control mice injected with irrelevant antigen-stimulated Thy-1.2+ T cells, only the occasional Thy-1.2+ T cell could be demonstrated up to 14 dpt. This is the first study showing unequivocally the presence of MBP-stimulated, adoptively transferred T cells within the CNS of recipients throughout the course of EAE, particularly during later relapsing stages. These results indicate that the persistent presence of antigen-specific T cells may be required for the recruitment of non-CNS antigen-responsive immune cells.

Based on previous observations showing that inflammatory episodes in the central nervous system (CNS) of SJL/J mice with adoptively transferred experimental allergic encephalomyelitis (EAE) are associated with a concomitant upregulation of adhesion-related molecules around CNS blood vessels, the present study was undertaken to block the development of EAE with injections of monoclonal antibodies (mAbs) to two different adhesion molecules. The mAbs selected were directed against intercellular adhesion molecule-1 (ICAM-1) and lymphocyte function-associated antigen-1 (LFA-1) and were administered at different doses (50 micrograms-1 mg), as single and multiple injections, and at various time points post-transfer of myelin basic protein-specific lymphocytes. Although one group of EAE mice given alpha LFA-1 displayed adverse effects after treatment, on the whole, neither mAb had a statistically significant effect on the outcome of EAE in this murine model. There was, however, down-regulation in the CNS of the respective adhesion molecules after treatment. Whether the lack of beneficial effect was related to the stage of EAE at which the mAbs were administered, remains to be proven. This is the first report suggesting that alpha ICAM-1 and alpha LFA-1 mAbs might have opposite effects (i.e. ameliorating or worsening) upon murine EAE and the different effect of alpha LFA-1 might be related to this molecule being involved in more cell signalling mechanisms than ICAM-1.

It had been demonstrated previously that the administration of transforming growth factor-beta 1 (TGF-beta 1) reduced the clinical severity of experimental allergic encephalomyelitis (EAE). Treatment with the related immunosuppressive molecule, TGF-beta 2, resulted in similar inhibition of T cell activation and proliferation in vitro. Long-term treatment was effective in reducing clinical severity of EAE and the number of relapses in mice receiving either myelin basic protein- or peptide-91-103-specific T cell lines. When examined histologically, mice that had received TGF-beta 2 demonstrated significantly less inflammation and demyelination in the central nervous system. Examination of other organs demonstrated no pathology or deleterious side effects from long-term TGF-beta 2 therapy. These findings have relevance for the use of TGF-beta 2 as a therapeutic agent for the human demyelinating disease, multiple sclerosis.

Supernates from myelin basic protein (MBP)-reactive T cell lines have been tested by a battery of assays for cytotoxicity against oligodendrocytes in vitro. All supernates tested demonstrated cytotoxic activity in both a dose- and time-dependent manner that ranged from 29.1% to 55.8% after 48 h incubation. Oligodendrocyte cytotoxicity mediated by MBP-reactive T cell lines was not antigen specific since lines reactive with purified protein derivative (PPD) of tuberculin showed similar cytotoxicity. Supernates cytotoxic to oligodendrocytes were equally effective against syngeneic and non-syngeneic target cells, but had no effect upon astrocytes. Neutralization studies revealed that oligodendrocyte cytotoxicity mediated by MBP-reactive T cell supernates could only be partially attributed to lymphotoxin/tumor necrosis factor activity, and was not associated with perforin, serine proteinase or N-methyl-D-aspartate (NMDA) receptor agonists.

The expression of two vascular addressins, adhesion molecules implicated in lymphocyte traffic via high endothelial venules (HEV) within lymph node and mucosal tissues, and of an HEV differentiation antigen (Ag) has been followed immunocytochemically in the central nervous system (CNS) of SJL mice at different stages of adoptively-transferred, chronic relapsing experimental autoimmune encephalomyelitis (EAE). Monoclonal antibody (mAb), MECA-325, which defines an HEV cell differentiation Ag generally associated with vessels involved in lymphocyte traffic, gave consistently high levels of expression on and around blood vessels within spinal cord lesions during periods of inflammation (acute onset and relapses). Two mAbs, MECA-89 and MECA-367, both recognizing the same mucosal addressin showed an affinity for endothelial cells and some astrocytes but only in lesions from animals displaying relapses. MECA-79, an mAb against a peripheral lymph node vascular addressin, showed no CNS staining whatsoever. All four antibodies gave uniformly positive staining on control lymphoid tissue. Since some stages of EAE appeared to be associated with the expression of different addressins, the possibility of separate roles for these distinct molecules should be considered.

Adoptively-transferred experimental allergic encephalomyelitis (EAE) was induced in the SWR (H-2q) strain of mouse using lymph node cells, spleen cells, or cell lines sensitized to whole myelin basic protein (MBP). With the aid of synthetic peptides, the minimal encephalitogenic epitope of MBP for the SWR strain was localized to amino acids 87-99 of the MBP molecule. The 87-99 sequence is also encephalitogenic for the SJL strain of mouse and the Lewis rat. EAE was induced with a protocol similar to that for the induction of EAE in the SJL strain with the exception that sublethal irradiation of recipients was necessary. Mice developed typical clinical and pathological EAE 6-14 days post-transfer of cells sensitized to either whole MBP or peptide 87-99, after which they remitted. No relapses were observed. Thus, adoptively transferred EAE can be induced in irradiated H-2q mice for which the encephalitogenic epitope is one of the nested encephalitogenic epitopes for SJL (H-2s) mice, namely residues 87-99.

In an attempt to understand the mechanisms underlying disease progression in adoptively transferred experimental allergic encephalomyelitis (EAE), and perhaps multiple sclerosis (MS), this study has examined the transfer of EAE serially from primary to secondary and tertiary recipients using myelin basic protein (MBP)-responsive lymphocytes. It was found that EAE could be serially transferred only when there was acute or relapsing disease activity in the donor animal. Cells from donors with quiescent disease did not transfer EAE. Autoradiographic attempts to locate primary adoptively transferred cells in the central nervous system of secondary and tertiary recipients were uniformly unsuccessful. These findings implicate the requirement of effector cell activation in the donor and the recruitment of augmenting autoimmune cells in lesion formation.

From previous studies on the induction and treatment of experimental autoimmune encephalomyelitis (EAE) in guinea pigs and mice, antibodies have been implicated during both demyelination and remyelination. In the present study, sera from guinea pigs with acute, chronic and myelin basic protein/galactocerebroside (MBP/GC)-treated chronic EAE were evaluated for the presence of anti-glial cell antibodies by immunocytochemical techniques. Antigen specificity was confirmed by enzyme-linked immunosorbent assay (ELISA) and Western blotting. The majority of sera from acute and chronic active EAE animals displayed intense labelling of astrocytes and only weak staining of oligodendrocytes when tested on sections of normal guinea pig brain tissue. In contrast, sera from animals with chronic EAE treated with MBP/GC gave strong labelling of oligodendrocytes and only minor staining of astrocytes. By immunoblotting, astrocyte staining was shown to be due to the presence of antiglial fibrillary acidic protein (GFAP) antibodies. The intense oligodendrocyte staining observed in sections reacted with sera from MBP/GC-treated guinea pigs corresponded well with high titers of serum anti-GC and anti-MBP antibodies measured by an ELISA. It was concluded that the presence of antibodies against astrocytes was possibly related to astrocytic antigens within the disease-inducing emulsion, at least during the initial phases of EAE, and not to their release from the central nervous system of affected animals.

The ultrastructure of dissociated rat sciatic nerve Schwann cells and fibroblasts has been examined and correlated at the morphologic and immunocytochemical levels. In agreement with previous studies, the two cell types were readily distinguishable morphologically on the basis of cell shape and size but no unique structural feature could be found. Schwann cells in this system essentially lacked basal laminae but did show some membrane specializations reminiscent of the in vivo situation. Fibroblasts were exceedingly rich in actin webs beneath the plasmalemma. Immunocytochemically, only Schwann cells stained with anti-Ran-1 antisera and fibroblasts only with anti-Thy-1. Both staining patterns were exclusively associated with the surface membrane. The findings provide a basis for future studies with this system to which neuronal elements may be added in an attempt to initiate myelinative events.

To test the hypothesis that T lymphocytes sensitized to central nervous system (CNS) antigens may quantitatively induce more demyelination in neural tissue than T cells sensitized to non-CNS antigens, we established T cell lines specific for myelin basic protein (MBP) or the purified protein derivative (PPD) of M. tuberculosis. The potential of T cells to cause myelin pathology was determined by measuring the activity of the myelin-associated enzyme 2'-3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) in organotypic cultures of syngeneic spinal cord after incubation with the T cell lines. The activity of CNPase in neural tissue has been shown to correlate positively with the amount and integrity of CNS myelin. Although both MBP- and PPD-specific T cells caused decreases in CNPase activity, the MBP line caused significantly greater and consistent changes. This finding indicates that T cell-mediated CNS demyelination may be comprised of CNS antigen-specific and CNS non-specific components, the former causing more pathology.

Lymphocyte responsiveness to oligodendrocytes has been examined with a modification of the antigen-reactive early T cell test in a total of 206 blood samples [104 multiple sclerosis (MS); 62 other neurological diseases (OND); 14 non-neurologic diseases; and 26 normals]. Oligodendrocyte reactivity was detectable more frequently in MS (49%) than in OND (23%) and normals (8%) but was absent from patients with non-neurologic diseases. Percentages of lymphocytes binding oligodendrocyte antigens were determined by direct and indirect immunofluorescence and immunocyto-adherence. Comparable results were obtained from all 3 systems. Percentages of oligodendrocyte antigen-binding lymphocytes were higher in MS than in OND and normals. With the present battery of techniques, lymphocyte responsiveness to oligodendrocytes has been shown to be more common in MS but since it is also found among OND, it is not MS-specific and most likely represents an epiphenomenon associated with white matter destruction.

Using monoclonal antibodies in combination with the PAP technique, total (T11+) T cells, helper-inducer (T4+) T cells, suppressor-cytotoxic (T8+) T cells and Ia+ cells (macrophages and B cells) were localized in frozen sections of multiple sclerosis (MS) lesions with varied disease activity. In acute MS, T11+, T4+, T8+ cells and Ia+ macrophages were found in large numbers throughout the lesion but were virtually absent from normal white matter. In active chronic MS lesions, the numbers of T11+, T4+ and T8+ cells increased from the center towards the edge of the lesion. T11+ and T4+ cells penetrated deeply into the normal-appearing white matter adjacent to the lesion, while T8+ cells were more confined to the lesion edge. Ia+ macrophages displayed a reverse distribution pattern to that of T cells. They showed the highest density in the lesion center and their numbers decreased slightly towards the lesion edge. Small numbers of T11+, T4+, T8+ and Ia+ cells were always present in normal white matter. In silent chronic MS lesions, the numbers of both T cells and Ia+ cells were significantly lower than in active chronic MS. While T11+ and T4+ cells were found throughout the central nervous system (CNS), T8+ cells were virtually absent from the lesion center. Ia+ macrophages were also present in small numbers throughout the CNS and, sometimes, showed some accumulation at the lesion edge. Thus, T cells and T cell subsets have been demonstrated to be involved in lesion pathogenesis in MS in that lesion progression was associated with T4+ cells while ongoing demyelination depended upon the presence of Ia+ macrophages.

It is postulated that the pathogenesis of demyelination in multiple sclerosis (MS) might lie in the cooperative effect of a T cell response against one myelin antigen (e.g. myelin basic protein--MBP) and a B cell response against a second myelin component which may act as a hapten or a carrier for the primary antigen. The hypothesis is based upon recent experiments in guinea pigs in which the encephalitogenicity of MBP was enhanced by the myelin glycolipid, galactocerebroside. This pathogenetic mechanism might be analogous to antibody-dependent, cell-mediated demyelination. Based upon this assumption, therapeutic trials in MS should take into consideration the possibility that instead of MBP alone, MBP might be more effective in combination with a lipid hapten.

Myelinated cultures of embryonic SJL/J mouse spinal cord tissue have been tested for their ability to express class II major histocompatibility complex (MHC) (Ia) molecules in the presence of two recombinant cytokines, mouse interferon-gamma (IFN-gamma) and human tumor necrosis factor-alpha (TNF-alpha). Both cytokines up-regulated class II MHC in a dose-dependent manner and interacted synergistically to increase Ia when applied together. The principal cell type involved were glial fibrillary acidic protein-positive astrocytes along the surface of the cultures. Immunocytochemistry in combination with light microscope examination of whole mounts and epoxy sections as well as electron microscopy, were used to assess the phenomena. These findings add further weight to previous observations on astrocytes being capable of serving as antigen-presenting cells within the central nervous system.

Epidural visual evoked potentials (VEP) were used to study the role of cytokines in the induction of pathophysiologic changes associated with inflammation in the central nervous system (CNS) of the rabbit. In normal rabbits, intraocular injection of human recombinant interferon-gamma (IFN-gamma) and tumor necrosis factor (TNF) increased the peak latency of the cortical VEP by more than 2 ms within 3 h of injection; equal volume injections of control substances had no effect. Alterations in conduction induced by IFN-gamma and TNF reversed within 24 h and could be reinduced by reinjection. Intraocular injection of recombinant human interleukin-1 beta (IL-1) induced a more progressive delay in conduction that peaked 24 h after intraocular challenge and reversed over the ensuing 48 h. Pathologic examination of the tissues indicated that the primary effect of these cytokines is on the vasculature and induces changes associated with inflammation. The results suggest that the acute reversible effects of cytokines on CNS function are associated with vascular events; further they support the sensitivity of the 'rabbit eye model' for studies on the pathophysiologic effect of inflammatory mediators on the CNS in vivo.

Previous work from this laboratory has shown that resistance to acute experimental autoimmune encephalomyelitis (EAE) correlates with disease-specific, antigen-induced suppression of blastogenesis in vitro. We now report that this suppression in vitro also occurs during remissions in animals with chronic-relapsing EAE. Hartley strain guinea pigs were injected with an homogenate of guinea pig spinal cord in complete Freund's adjuvant (CFA) to induce EAE or, for control purposes, with CFA alone. Animals injected with spinal cord homogenate developed EAE. Susceptible animals displayed up to 3 exacerbations over 4-5 months. Spleen cells and nervous tissue were sampled from different animals during and after each exacerbation. Gross examination of nervous tissue revealed plaques that at the light microscope level were characteristic of chronic-relapsing EAE. Lymphocyte transformation assays using the T-cell mitogen concanavalin A (Con A), guinea pig myelin basic protein (BP), the purified protein derivative of M. tuberculosis (PPD) and histone proteins were conducted. Results of these assays showed that in spleen cells from animals sampled during remissions, BP suppressed the Con A response. Similar suppression was not observed with spleen cells from animals in exacerbation. This suppression depended upon the presence of adherent cells. Neither PPD nor histone proteins suppressed the Con A response. Thus, an immunologic mechanism, similar to that observed in Hartley guinea pigs resistant to acute EAE, is also found during remissions in the chronic-relapsing form of this disease suggesting that both resistance and remission are mediated by an antigen-induced suppressor mechanism.

One-micron plastic sections of spinal cords from SJL/J mice with chronic relapsing experimental allergic encephalomyelitis (EAE) were reacted immunocytochemically with antiserum to myelin basic protein and myelin-associated glycoprotein. The distribution of myelin basic protein and myelin-associated glycoprotein in myelin sheaths was compared in acute and chronic areas of demyelination. No difference in the size of the lesion was seen with the two antisera. Myelin-associated glycoprotein was seen periaxonally in both normal myelin sheaths and sheaths which showed extensive splitting and ballooning as seen with toluidine blue stain and myelin basic protein antiserum. At least at the level of the light microscope, myelin basic protein antiserum gave intense staining of myelin while antiserum to myelin-associated glycoprotein showed little or no affinity to stain the myelin sheath itself, in contrast to other recent electron microscope observations. A few myelin basic protein or myelin-associated glycoprotein-containing oligodendrocytes were seen in lesion areas and remyelination by oligodendrocytes was rare. These observations are in agreement with findings from other models of EAE and multiple sclerosis where a primary loss of myelin has been implicated.

Chronic relapsing experimental allergic encephalomyelitis (CREAE) was induced in SJL mice following the adoptive transfer of a T cell line derived from mice immunized with a synthetic peptide corresponding to residues 89-100 of the guinea pig myelin basic protein (MBP) molecule. This cell line proliferated to both the peptide and MBP and induced CREAE characterized by a series of relapses with eventual stabilization. Central nervous system (CNS) inflammation and demyelination were prominent neuropathologic features of both the acute and relapsing phase of the disease. The chronic phase was characterized by CNS lesions containing chronically demyelinated fibers with remyelination and some fiber drop-out, but little inflammation. The induction of CREAE in SJL mice by a cell line specific for residues 89-100 demonstrates that T cell recognition of this epitope is required for successful disease induction in this strain of mouse.

The expression of major histocompatibility complex (MHC) molecules by oligodendrocytes has been proposed as evidence for their involvement in the multiple sclerosis (MS) lesion although the literature on the subject is controversial and based largely upon observations in vitro. With a modified immunocytochemical procedure on 1 micron epoxy sections, the present study has examined the expression of class II MHC molecules (Ia) on cells within actively demyelinating lesions in a central nervous system biopsy from a case of acute MS. White Ia was readily demonstrable on microglial cells and astrocytes, it was never detected on adjacent surviving oligodendrocytes. Unexpectedly, in parallel sections, the oligodendrocytes stained positively for myelin-associated glycoprotein, a marker for immature oligodendrocytes. The unequivocal lack of Ia expression by oligodendrocytes in MS makes it unlikely that they serve as immunomodulators in lesion pathogenesis.

Experimental autoimmune (allergic) encephalomyelitis (EAE) was induced in Strain 2 guinea pigs, a strain usually regarded as resistant to EAE. The development of disease in groups of juvenile male or female Strain 2/N guinea pigs was irregular with clinical EAE manifesting only in some groups of females. All animals examined morphologically (12 females and 9 males), between 3 and 18 months postinoculation, showed extensive changes in the central nervous system, although 12 of these had displayed no neurologic signs. This silent central nervous system disease, reminiscent of similar phenomena in man, indicates that Strain 2/N is fully competent immunologically at the level of the target organ.

Lymphocytes from the central nervous system (CNS) of animals with acute experimental autoimmune encephalomyelitis (EAE) have been isolated and characterized. The lymphocytes were separated from Lewis rats which were injected either with an emulsion of myelin basic protein (BP) emulsified in complete Freund's adjuvant (CFA) to cause EAE or with CFA alone as a control. Using density gradient centrifugation, from 9 days post-inoculation (d.p.i.) (before clinical signs appear), to 19 d.p.i. (after signs abate), lymphocytes were recovered from the spinal cords and popliteal lymph nodes of BP-injected animals. Lymphocyte cell number, phenotype, and antigen specificity were determined. Results show that the onset of clinical signs correlated with lymphocyte influx into the CNS. A clinical index of 1 was associated with less than 10(6) cells per gram of CNS wet weight (cells/g CNS) while animals with a clinical index of 4 had more than 15 X 10(6) cells/g CNS. During remission, when only minor residual neurologic signs were evident, significant numbers of lymphocytes (greater than 10(7) cells/g CNS) could still be isolated. In contrast, no lymphocytes were obtained from control CNS tissue. The phenotype of the recovered cells was predominantly of the helper/inducer T cell subset (greater than 40%). Although the percentages of these cells in the CNS were increased when compared to the lymph nodes, I-A expression on CNS-isolated lymphocytes showed the most significant increase with disease progression. Recovered lymphocytes responded to both BP and CFA-related antigens indicating that both CNS-specific and CNS-non-specific inflammatory cells were present in the exudate.

Monoclonal anti-human T cell antibodies were tested by a modified PAP technique on frozen sections of human central nervous system (CNS) tissue from inflammatory and non-inflammatory conditions. It was found that whole T cells and T cell subsets--T4+ (helper-inducer) and T8+ (suppressor/cytotoxic) T cells--could be differentiated specifically from other mononuclear cells and that these markers did not cross-react with human CNS tissue elements, particularly oligodendrocytes, under the same conditions. The lack of cross-reactivity between monoclonal antibodies to T cells and oligodendrocytes was further confirmed by double-labelling with an anti-galactocerebroside serum. It is concluded that in inflammatory conditions like multiple sclerosis, monoclonal antibodies promise considerable elucidation of immunopathogenetic events.

Using a monoclonal antibody in combination with the 4-step modified peroxidase-antiperoxidase (PAP) technique, Ia expression was demonstrated on endothelial cells and astrocytes in MS lesions of different ages. On endothelial cells, Ia antigen was found most frequently in grey and white matter parenchyma of acute MS brain and showed lower, comparable numbers in active and silent chronic MS brain tissue. Ia+ astrocytes were most numerous in acute MS lesions. In active chronic MS, Ia+ astrocytes predominated at the lesion edge, where they frequently displayed an atypical, rounded configuration. Positive astrocytes showed somewhat lower numbers within the lesion center and in normal white matter close to the lesion edge. Their frequency was significantly lower in normal white matter remote from the lesion. In silent chronic MS, Ia+ astrocyte processes were detectable only within the lesion center. Grey matter astrocytes displayed no staining with anti-Ia antibody. In normal brain tissue, no Ia antigen could be detected. The presence of Ia molecules on some endothelial cells and astrocytes in MS brain tissue suggests a role in antigen presentation perhaps relevant to the initiation and perpetuation, respectively, of the inflammatory process.