D. Lehmann

Chronic-relapsing experimental autoimmune encephalomyelitis (CR-EAE): treatment and induction of tolerance, with high dose cyclophosphamide followed by syngeneic bone marrow transplantation

We examined the effect of acute immunosuppression with high dose cyclophosphamide (CY), followed by syngeneic T-cell-depleted bone marrow transplantation (SBMT) on chronic-relapsing autoimmune encephalomyelitis (CR-EAE) induced in SJL/J mice by immunization with mouse spinal cord homogenate (MSCH) in adjuvant. Treatment of mice on day 9 post immunization, before the appearance of clinical signs of the disease, delayed the onset of paralysis, but did not affect its clinical course. Treatment on day 2-3 after the first clinical signs led to complete regression of the disease. During a period of 3 months, only one of the 15 mice treated after the the onset of CR-EAE relapsed, as compared to a total of 21 relapses in the 15 untreated animals. A rechallenge with MSCH in adjuvant on day 78 after immunization induced a severe relapse in all untreated mice, with 78% mortality; in contrast, only 25% of mice treated with CY and SBMT relapsed when similarly rechallenged. Lymphocytes from mice treated with CY and SBMT showed reduced in vitro proliferative responses to myelin basic protein (GMBP) and PPD, even after the rechallenge with MSCH. Our results show that high dose CY for elimination of immunocompetent lymphocytes, followed by SBMT rescue, suppresses CR-EAE and induces tolerance to the immunizing antigens. These results may encourage attempts to apply a similar therapeutic principle in life-threatening human neurological autoimmune diseases.

γ-Irradiation enhances apoptosis induced by cannabidiol, a non-psychotropic cannabinoid, in cultured HL-60 myeloblastic leukemia cells

Two non-psychotropic cannabinoids, cannabidiol (CBD) and cannabidiol-dimethylheptyl (CBD-DMH), induced apoptosis in a human acute myeloid leukemia (AML) HL-60 cell line. Apoptosis was determined by staining with bisBenzimide and propidium iodide. A dose dependent increase of apoptosis was noted, reaching 61 and 43% with 8 μg/ml CBD and 15 μg/ml CBD-DMH, respectively, after a 24 h treatment. Prior exposure of the cells to γ-irradiation (800 cGy) markedly enhanced apoptosis, reaching values of 93 and 95%, respectively. Human monocytes from normal individuals were resistant to either cannabinoids or γ-irradiation. Caspase-3 activation was observed after the cannabinoid treatment, and may represent a mechanism for the apoptosis. Our data suggest a possible new approach to treatment of AML.

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.

The Role of Skin-Derived Dendritic Cells in CD8+ T Cell Priming Following Immunization with Lentivectors

Although skin dendritic cells (DCs) have been shown to directly present Ag to CD8+ T cells after intradermal immunization with lentivectors, the contribution of the different skin DC subsets to this process remains unclear. Using langerin-diphtheria toxin receptor transgenic mice we demonstrated that ablation of langerhans cells and langerin-expressing positive dermal DCs (Ln+dDCs) did not interfere with the generation of CD8+ T cells by lentiviral vectors. Consistent with these findings, the absence of langerhans cells and Ln+dDCs did not hamper the presentation level of lentiviral-derived Ag by skin DCs in vitro. We further demonstrated that only dDCs and Ln+dDCs were capable of presenting Ag, however, the number of dDCs migrating to the draining lymph nodes was 6-fold higher than that of Ln+dDCs. To study how the duration of DC migration influences CD8+ T cell responses, we analyzed the kinetics of Ag expression at the injection site and manipulated DC migration by excising the injected skin at various times after immunization. A low level of Ag expression was seen 1 wk after the immunization; peaked during week 2, and was considerably cleared by week 3 via a perforin-dependent fas-independent mechanism. Removing the injection site 3 or 5 d, but not 10 d, after the immunization, resulted in a reduced CD8+ T cell response. These findings suggest that dDCs are the main APCs active after intradermal lentiviral-mediated immunization, and migration of dDCs in the initial 10-d period postimmunization is required for optimal CD8+ T cell induction.

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