R. Mizrachi-Koll

Acute/relapsing experimental autoimmune encephalomyelitis: Induction of long lasting, antigen-specific tolerance by syngeneic bone marrow transplantation

Experimental autoimmune encephalomyelitis (EAE) is an inducible autoimmune disease widely used as a model of the acute/relapsing stage of multiple sclerosis. We have previously shown that treatment of EAE-mice with high doses of cyclophosphamide (CY) (350 mg kg), followed by syngeneic bone marrow transplantation (SBMT), completely abrogates the clinical paralytic signs and even prevents the appearance of new relapses in the chronic-relapsing model of the disease. In the present study we examined whether this treatment protocol induces long term tolerance and whether this tolerance is antigen-specific. EAE was induced by immunization with spinal cord homogenate (MSCH) in complete Freunds adjuvant (CFA). The treatment with CY and SBMT was performed on day 6 post immunization. Treated and untreated mice were rechallenged with MSCH, or a non-relevant antigen (OVA) in CFA at various stages after the first paralytic attack. In contrast to previous data showing that animals recovering from acute EAE are usually refractory to re-induction of the disease, repeated injections of MSCH at different sites from the initial immunization, followed by i.v. injection of inactivated Bordetella bacteria, 2, 4 and 6 months after the initial EAE-induction, caused a severe and usually lethal relapse in all the untreated, control animals. Mice treated with CY and SBMT were resistant to all rechallenges with the same encephalitogenic inoculum. Following the second rechallenge, peripheral lymph node cells were examined in vitro for their proliferative responses to myelin antigens or to OVA. Lymphocytes obtained from CY+SBMT treated mice did not proliferate in vitro in response to myelin basic protein (MBP), but proliferated against OVA, when immunized with this antigen, after SBMT. Adoptive transfer of lymphocytes from tolerant mice to naive recipients did not transfer resistance to EAE-induction. Our results indicate that high doses of CY, followed by SBMT, induce long term antigen-specific tolerance presumably by a mechanism of clonal deletion or anergy.

Immunomodulation of experimental autoimmune myasthenia gravis with linomide

Linomide, a synthetic immunomodulator, increases natural killer (NK) activity and markedly activates several lymphocyte populations in both experimental animals and humans. It has been shown to ameliorate the autoimmune manifestations of lupus-like disease in MRL/lpr mice and the clinical and pathological signs of acute and chronic-relapsing experimental autoimmune encephalomyelitis (EAE) in SJL/J mice. We examined the effect of linomide (100 mg/kg/day; administered in drinking water) on rabbits and rats with experimental autoimmune myasthenia gravis (EAMG). Following immunization with Torpedo acetylcholine receptor (AChR), all control rabbits developed clinical signs of severe weakness and exhibited a decrement of muscle action potential upon repetitive stimulation. In contrast, mild signs of weakness appeared in only two of five linomide-treated rabbits, with EMG borderline positive in one of them. Booster immunization with Torpedo AChR induced severe relapse and death in two EAMG control rabbits, whereas the two linomide-treated animals remained free of myasthenic symptoms. The serum level of antibodies against both Torpedo and rat AChR were markedly suppressed in the linomide-treated animals. Similar inhibition of clinical signs of EAMG was observed in the EAMG rat model. Furthermore, the in vitro proliferative response of lymph node cells to Torpedo AChR and the purified protein derivative of Mycobacterium tuberculosis was significantly lower in the linomide-treated EAMG rats than in the controls. Linomide may constitute a new immunomodulating agent for the treatment of myasthenia gravis.

Immunomodulation of autoimmunity by linomide: inhibition of antigen presentation through down regulation of macrophage activity in the model of experimental autoimmune encephalomyelitis

Linomide (quinoline-3-carboxamide, LS-2616), a synthetic immunomodulator, protects animals against a variety of experimental autoimmune diseases. In experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis (MS), linomide blocks both the clinical and histological signs of the disease, without inducing generalized immunosuppression. In the first clinical trial in patients with MS, linomide was shown to inhibit the progression of the disease. In the present study we investigated several aspects of the mechanisms of action of this immunomodulator. We found that linomide can inhibit acute EAE even when given as pretreatment, prior to induction of disease (days - 10 to 0). This inhibitory effect was reversed by adoptive transfer of naive spleen cells. A short course (7 days) of linomide treatment also inhibited EAE, especially when administered immediately after disease induction. Spleen cells from linomide-treated mice failed to present myelin antigens to T-cell lines in vitro. The defective antigen presentation was normalized by anti-oxidants such as 2-mercaptoethanol. The proportion of Mac1+ cells in the spleens of linomide-treated mice was significantly reduced and macrophage growth was inhibited in long term cultures of spleen cells derived from linomide-treated animals. Our findings suggest that the effect of linomide on EAE may be attributed, at least in part, to inactivation of antigen presenting cells, possibly following a short period of over-stimulation and increased oxidant production. This mechanism may play a universal role in the regulation of autoimmune reactivity and merits further investigation.

LINOMIDE DOWNREGULATES AUTOIMMUNITY THROUGH INDUCTION OF TH2 CYTOKINE PRODUCTION BY LYMPHOCYTES

Linomide is a synthetic immunomodulator that has been shown to protect animals against a wide range of spontaneously developing or induced autoimmune diseases. We have previously reported that Linomide blocks both the clinical and the histopathological manifestations of experimental autoimmune encephalomyelitis (EAE) in various animal models. In this study, in an effort to elucidate the mechanisms by which Linomide suppresses EAE, and autoimmunity in general, we investigated the in vivo effects of this drug on the TH1/TH2 lymphocyte balance, which is important for the induction or inhibition of autoireactivity. Naive SJL/J mice were treated orally for 15 days with Linomide (80 mg/kg/day). Spleen cells were obtained at various time points during the treatment period and were stimulated in vitro with concanavalin A. Interleukins IL-4, IL-10 and IL-12, transforming growth factor-beta (TGFbeta) and interferon-gamma (IFNgamma) cytokine production was evaluated both by means of detection of the cytokines in the medium (by ELISA technique) and by detection of the cytokine mRNA production, using a semiquantitative reverse transcriptase polymerase chain reaction method. A significant upregulation of IL-4, IL-10 and TGFbeta was observed following treatment with Linomide, which peaked at day 10 (IL-10) or day 15 (IL-4). On the other hand, IL-12 and IFNgamma production were either unchanged or decreased. It seems therefore that Linomide induces in vivo a shift towards TH2 lymphocytes which may be one of the mechanisms of downregulation of the autoimmune reactivity in EAE. Our observations indicate that downregulation of TH1 cytokines (especially IL-12) and enhancement of TH2 cytokine production may play an important role in the control of T-cell-mediated autoimmunity. These data may contribute to the design of new immunomodulating treatments for a group of autoimmune diseases.

CD44 variant DNA vaccination with virtual lymph node ameliorates experimental autoimmune encephalomyelitis through the induction of apoptosis.

Standard CD44 (CD44s) and its alternatively spliced variants (CD44v) were found to be associated with the metastatic potential of tumor cells, and with cell migration of autoimmune inflammatory cells, including cells involved in experimental autoimmune encephalomyelitis (EAE). The aim of the present study was to evaluate whether induction of anti-CD44 immune reactivity, through cDNA vaccination could down-regulate EAE. Our vaccination technique involved the insertion of CD44s or CD44v cDNA into a silicone tube filled with 2.5 cm long segment of hydroxylated-polyvinyl acetate wound dressing sponge (forming a virtual lymph node) which was implanted under the skin of SJL/J mice immunized with myelin antigens for EAE induction. Animals vaccinated with CD44v cDNA developed significantly less severe EAE when compared with sham vaccinated animals or animals vaccinated with CD44s cDNA. The in vitro proliferation of lymphocytes was preserved regarding myelin antigens and mitogens. Histopathological examinations revealed a significant reduction of EAE lesions and enhanced apoptosis in central nervous system (CNS)-infiltrating cells of the successfully vaccinated animals. Such methods of cDNA vaccination with CD44 could be applicable in inflammatory CNS diseases, like multiple sclerosis.

Experimental allergic encephalomyelitis is suppressed with a nitric oxide synthase inhibitor

Introduction: Because T cells in the cerobrospinal fluid (CSF) located close to the lesion may have the potential to cause the pathogencsis of multiple sclerosis (MS) mote than T cells in the peripheral blood, we investigated T cell lines that responded to myelin basic protein (MBP) from the CSF of MS patients. Materials and Methods: CSF cells (2.2xl0~/patient) from eight MS patients were cultured (10S/well) with 10 s of irradiate autologoos peripheral blood mononaclear cells, MBP (20pg/ml) and IL-2 for 20 days. Then, each CSF T cell line was tested for reactivity to MBP and MBP peptides (MBP 1-20, 11-30, 21-40, 31-50, 41-60, 51-70. 61-82, 71-92, 84-I02, 93-I12, 113-132, 124-142, 143-168). Results: A total of 14 MBP lines were obtained from 5 out of 8 subjects (0 to 5 MBP lines/subjec 0. The average incidence of MBP lines out of all starting culture wells and the average frequency of MBP reactive T cells out of the total number of starting CSF cells were 9.1_+11.5% a.d 0.7_+1.1x10 ~, respectively. Of these 14 T cell lines, 5 lines responded to MBP 84-102 and 5 other lines showed only MBP reactivity. In one subject, 3 of all 5 MBP lines responded to MBP 84-102. Furthermore, an MBP 84-102 reactive line was noted in 2 other subjects. Regarding another epitopes, two MBP 31-50, one 41-60 and one 51-70 reactive lines were established from different subjects. Conclusion: The finding that the frequency of MBP reactive T cells from the CSF was higher than that from the peripheral blood (4.1+1.8x10 -~) in MS (Can J Nenrol Sci 20 suppl 4, S133) suggests that MBP reactive T cells in the CSF may play an important rule in the pathogenesis of MS.

Successful treatment of chronic-relapsing experimental autoimmune encephalomyelitis with linomide (LS-2616)

Reel C. van d e r Veen, John L. Trot te r , Jud i th A. Kapp , Washin~un University School of Medicine, St. Louis, M e 63110, USA Myelin proteolipid protein (PLP) has one recognized epitope wi*.hin the 139 -151 amino acid sequence (PLP-EP), that is encephalitogenic for SJL mice. In the current study, the proees~ing of P IP by different antigen-presenting cells (APC) was examined. In order to study whether PIP requires processing before its presentation by APC, PiP-pulsed and fixed APC were shown to stinmlate PiP-specific T cells. However, the addition of P IP to unpl~.lsed, fixed APC resulted in the absence ofT-cell stimulation, while the viability of these fixed PEC to bind antigenic peptide and efficiently present it to T cells was demonstrated by their ability to use a synthetic peptide for the stimulation of T cells. In order to study possible processing differences among APC subsets, spleen cells were fractionated by adherence to plastic, and their respective APC a:tivities were studied separately. Although both adherent and non-adherent spleen cells stimulated PLP-specific T-cell lines, the non-adherent APC were unable to stimulate an encephalitogenic T-cell clone with PIP. The specificity of the limited APC ability by non-adherent spleen cells was indicated by similar results us;.ng a second T-cell clone with specificity for the encephalitogenic pepfide. In contrast to these PLP-EP specific clones, a T-cell clone specific for a separate, unidentified epitope on PIP was stimulated by non-adherent APC efficiently. In contrast, stimulation of PLP-EP specific T-cell clones by nowadherent APC did occur when the synthetic peptide instead of intact P IP was used as antigen, suggesting a defect in PIP processing by the non-adherent APC. Further fractionation of the irradiated, non-adherent spleen cells demonstrated that their activity was limited to a nonB, and non-T cell subfraction. These results indicate that a subpopulation of spleen APC is unable to process PIP efficiently, and more specifically, may be unable to process a fragment containing the 139! 51 sequence in PIP, while other fragments of P IP can be processed and suby, e~luently presented efficiently. 159