Ling-Yun Xu

Laquinimod (ABR-215062) suppresses the development of experimental autoimmune encephalomyelitis, modulates the Th1/Th2 balance and induces the Th3 cytokine TGF-β in Lewis rats

The new orally active drug laquinimod (ABR-215062) was evaluated in experimental autoimmune encephalomyelitis (EAE) in the Lewis rat. EAE shares important immunological and clinical features with multiple sclerosis (MS). Doses of 16, 1.6 and 0.16 mg/kg/day laquinimod dose-dependently inhibited disease and showed better disease inhibitory effects as compared to roquinimex (Linomide). Furthermore, laquinimod inhibited the inflammation of both CD4+ T cells and macrophages into central nervous tissues, i.e. the spinal cord. It also changed the cytokine balance in favour of TH2/TH3 cytokines IL-4, IL-10 and TGF-beta. Laquinimod therefore represents a new orally active immunoregulatory drug without general immunosuppressive properties with a potential for the treatment of severe autoimmune diseases like MS.

Autoantigen‐pulsed dendritic cells induce tolerance to experimental allergic encephalomyelitis (EAE) in Lewis rats

Dendritic cells (DC) can modulate the nature of immune responses in a stimulatory or tolerogenic fashion. Great attention has been given to the induction of immunity to tumour and infection. In this study, bone marrow‐derived DC from healthy Lewis rats were pulsed in vitro with encephalitogenic myelin basic protein peptide 68–86 (MBP 68–86), and injected subcutaneously (1 × 106/rat) into normal Lewis rats. Upon observation of the rats pretreated in this way for 4 weeks, when no clinical signs of EAE occurred, these rats were immunized with MBP 68–86 and Freunds complete adjuvant. The pretreated rats failed to develop clinical EAE. This tolerance was associated with augmented proliferative responses, interferon‐gamma secretion, inducible nitric oxide synthase (iNOS) expression and NO production. The frequency of apoptotic cells was increased in the rats receiving MBP 68–86‐pulsed DC compared with unpulsed control DC. Few infiltrating inflammatory cells were observed in spinal cord sections from rats that had received MBP 68–86‐pulsed DC. The data are compatible with the interpretation that MBP 68–86‐pulsed DC induce tolerance to EAE possibly through up‐regulation of iNOS expression and NO production, which mediate cell apoptosis, thereby reducing infiltration of inflammatory cells within the central nervous system.

Adherent dendritic cells expressing high levels of interleukin-10 and low levels of interleukin-12 induce antigen-specific tolerance to experimental autoimmune encephalomyelitis

We have previously shown that tolerance can be induced against acute experimental autoimmune encephalomyelitis (EAE) in Lewis rats by bone marrow‐derived dendritic cells (DC) that have been pulsed in vitro with encephalitogenic myelin basic protein peptide 68–86 (MBP 68–86), and injected subcutaneously into healthy rats prior to immunization with MBP 68–86 plus complete Freunds adjuvant. To elucidate better the properties of tolerogenic DC, we here compared plastic‐adherent DC with floating, non‐adherent DC, which were cultured for 7 days in the presence of granulocyte–macrophage colony‐stimulating factor plus interleukin‐4 (IL‐4). Adherent DC expressed high levels of IL‐10 mRNA and protein, and low levels of IL‐12 mRNA and showed high expression of CD54 compared with floating DC. Proliferation, nitrite concentration and capacity for antigen presentation were lower in adherent DC than in floating DC. There were no differences between adherent and floating DC regarding expression of CD11c, OX62, major histocompatibility complex class II, CD80, or CD86. Most importantly, we observed that adherent DC induced tolerance to EAE in vivo when injected subcutaneously into Lewis rats prior to immunization, while floating DC did not. Adherent DC‐mediated tolerance to EAE was associated with augmented proliferation, nitric oxide production and frequency of apoptotic cells as well as with up‐regulation of transforming growth factor‐β (TGF‐β) ‐expressing cells in T‐cell areas of lymph nodes. Tolerance induction by adherent DC seems to be related to a nitric oxide–apoptosis pathway and to up‐regulation of TGF‐β‐expressing cells.

Mechanisms of recovery from experimental allergic encephalomyelitis induced with myelin basic protein peptide 68-86 in Lewis rats: a role for dendritic cells in inducing apoptosis of CD4+ T cells.

Spontaneous remission of experimental allergic encephalomyelitis (EAE) is usually associated with prominent apoptosis. The mechanisms behind apoptosis are unknown. We examined the functions of dendritic cells (DC) from Lewis rats with EAE induced by immunization with myelin basic protein peptide 68-86 (MBP68 - - 86). Recovery from EAE was associated with three major functional changes of freshly prepared DC: (1) elevated proliferation, (2) increased nitric oxide (NO) production, and (3) augmented IFN-gamma secretion. In Freunds complete adjuvant (FCA)-immunized control rats, no increase of proliferation, NO production or IFN-gamma secretion was observed on day 21 post-immunization (p.i.), i.e., recovery from EAE. In vitro effects of IFN-gamma, TNF-alpha, TGF-beta1, IL-4 and IL-10 on DC were examined. IFN-gamma enhanced proliferation and NO production by DC, while TNF-alpha and IL-4 induced only slight DC proliferation. DC from recovering EAE rats (day 21 p.i.) suppressed MBP68 - - 86-induced T cell proliferation compared to DC obtained at other time points in EAE and FCA-immunized rats. DC-derived NO induced apoptosis of CD4+ T cells, thereby inhibiting autoreactive T cell responses. Besides IFN-gamma stimulation, NO production by DC was mainly induced in an antigen-dependent manner when DC were co-cultured with T cells. The results suggest that spontaneous recovery from EAE is associated with augmented DC functions. Overproduction of NO by DC results in apoptosis of autoreactive CD4+ T cells, thereby decreasing autoreactive T cell reactivities. The existence of such a NO negative feedback loop may contribute to remission of EAE.

SIN-1, a Nitric Oxide Donor, Ameliorates Experimental Allergic Encephalomyelitis in Lewis Rats in the Incipient Phase: The Importance of the Time Window

NO is involved in the regulation of immune responses. The role of NO in the pathogenesis of experimental allergic encephalomyelitis (EAE) is controversial. In this study, 3-morpholinosydnonimine (SIN-1), an NO donor, was administered to Lewis rats on days 5–7 postimmunization, i.e., during the incipient phase of EAE. SIN-1 reduced clinical signs of EAE compared with those in PBS-treated control rats and was accompanied by reduced ED1+ macrophages and CD4+ T cell infiltration within the CNS. Blood mononuclear cells (MNC) obtained on day 14 postimmunization revealed that SIN-1 administration enhanced NO and IFN-γ production by blood MNC and suppressed Ag- and mitogen-induced proliferative responses. MHC class II, B7-1 and B7-2 were down-regulated in SIN-1-treated EAE rats. Simultaneously, frequencies of apoptotic cells among blood MNC were increased. In vivo, SIN-1 is likely to behave as an NO donor. Administration of SIN-1 induced NO production, but did not affect superoxide and peroxynitrite formation. Enhanced NO production during the priming phase of EAE thus promotes apoptosis, down-regulates disease-promoting immune reactivities, and ameliorates clinical EAE, mainly through SIN-1-derived NO, without depending on NO synthase.

Dendritic cell-derived nitric oxide is involved in IL-4-induced suppression of experimental allergic encephalomyelitis (EAE) in Lewis rats

Cytokines play a crucial role in initiating and perpetuating EAE, an animal model of multiple sclerosis (MS). A low dose of IL‐4, administered by the nasal route over 5 days (100 ng/rat per day) prior to immunization, improved clinical scores of EAE induced in Lewis rats with myelin basic protein (MBP) peptide 68–86 (MBP 68–86). We examined whether dendritic cells (DC) may have contributed to the amelioration of the disease process. These professional antigen‐presenting cells (APC) not only activate T cells, but also tolerize T cells to antigens, thereby minimizing autoimmune reactions. We found that IL‐4 administration enhanced proliferation of DC. In comparison with DC of PBS‐treated rats, DC from IL‐4‐treated rats secreted high levels of interferon‐gamma (IFN‐γ) and IL‐10. Nitric oxide (NO) production by DC was also strongly augmented in IL‐4‐treated rats. In vitro studies showed that IL‐4 stimulated DC expansion and that IFN‐γ enhanced NO production by DC. DC‐derived NO promoted apoptosis of autoreactive T cells. These results indicate that nasal administration of IL‐4 promotes activation of DC and induces production of IFN‐γ and IL‐10 by DC. IL‐10 suppresses antigen presentation by DC, while IFN‐γ induces NO production by DC which leads to apoptosis in autoreactive T cells. Such a DC‐derived negative feedback loop might contribute to the clinical improvement observed in EAE.

Bone marrow-derived dendritic cells from experimental allergic encephalomyelitis induce immune tolerance to EAE in Lewis rats

We have previously shown that dendritic cells (DC), upon being pulsed in vitro with encephalitogenic myelin basic protein peptide 68–86 (MBP 68–86) and injected subcutaneously (s.c.) back to healthy Lewis rats, transfer immune tolerance to experimental allergic encephalomyelitis (EAE) induced by immunization with MBP 68–86 and Freunds complete adjuvant (FCA). We here assumed that DC become pulsed in EAE rats, and that expansion in vitro of such ‘in vivo pulsed EAE‐DC’ might also have the capacity to induce immune tolerance to EAE, thereby eliminating the need for in vitro pulsing of DC with autoantigens which are still unknown in many autoimmune diseases in the human. In the present study, EAE‐DC were generated from bone marrow of Lewis rats, with EAE induced with MBP 68–86 + FCA, and expanded in vitro by culture with GM‐CSF and IL‐4. In comparison with DC from normal rats, EAE‐DC exhibited higher viability in the absence of growth factors, and presented specific antigen to naïve T cells in vitro. The DC derived from both EAE and healthy rats stimulated strong proliferation in an antigen‐independent manner, lasting for 4 weeks after DC were s.c. injected into healthy rats. During this time, injection of EAE‐DC did not induce clinical EAE. However, when these rats were immunized with MBP 68–86 + FCA, subsequent EAE was dramatically suppressed, and was associated with increased IFN‐γ expression, nitric oxide production, gradually reduced proliferation and cell apoptosis, compared with PBS‐injected control EAE rats. LPS‐treated DC did not induce tolerance, suggesting that the tolerance is mediated by an immature stage of DC. These observations support the hypothesis that EAE‐DC can transfer immune tolerance to EAE, thereby omitting the step of characterizing specific autoantigen. Omitting the step of loading DC with antigen not only eliminates the extremely complex procedure of defining pathogenically‐relevant autoantigens, but also avoids the risk of inducing immunogenicity of DC in the treatment of autoimmune diseases.

Suppression of ongoing experimental allergic encephalomyelitis (EAE)in Lewis rats: synergistic effects of myelin basic protein (MBP) peptide68–86 and IL‐4

Mucosal myelin autoantigen administration effectively prevented EAE, but mostly failed to treat ongoing EAE. Patients with multiple sclerosis (MS), for which EAE is considered an animal model, did not benefit from oral treatment with bovine myelin. We anticipated that autoantigen, administered together with a cytokine that counteracts Th1 cell responses, might ameliorate Th1‐driven autoimmune disease, and that nasal administration might considerably reduce the amounts of antigen + cytokine needed for treatment purposes. Lewis rats with EAE actively induced with myelin basic protein peptide (MBP 68–86) and Freunds complete adjuvant (FCA), received from day 7 post‐immunization, i.e. after T cell priming had occurred, 120 μg MBP 68–86 + 100 ng IL‐4 per rat per day for 5 consecutive days. These rats showed later onset, lower clinical scores, less body weight loss and shorter EAE duration compared with rats receiving MBP 68–86 or IL‐4 only, or PBS. EAE amelioration was associated with decreased infiltration of ED1+ macrophages and CD4+ T cells within the central nervous system, and with decreased interferon‐gamma (IFN‐γ) and tumour necrosis factor‐alpha (TNF‐α) and enhanced IL‐4, IL‐10 and transforming growth factor‐beta (TGF‐β) responses by lymph node cells. Simultaneous administration of encephalitogenic peptide + IL‐4 by the nasal route thus suppressed ongoing EAE and induced IL‐4, IL‐10 and TGF‐β‐related regulatory elements.

Limitation of nitric oxide production: cells from lymph node and spleen exhibit distinct difference in nitric oxide production.

Many types of cells in the immune system have been found to produce nitric oxide (NO), which performs multiplex functions. However, in myelin basic protein peptide 68-86 (MBP 68-86)-induced experimental autoimmune encephalomyelitis (EAE) in Lewis rats, we found that elevated NO production was generated from spleen cells (SC), not from lymph node cells (LNC). LNC expressed lower NO synthase 2 (NOS2) and produced lower levels of NO than SC upon MBP 68-86 stimulation. Expression of B7-1(CD80) and B7-2(CD86) molecules was much lower on LNC than on SC. Blocking of B7-1 or B7-2 ligation resulted in reduced NO production by SC. Unlike SC, LNC were resistant to interferon-gamma- or lipopolysaccharide-induced NO production. NO derived from SC suppressed cell proliferation and induced apoptosis in vitro. Addition of N(omega)-nitrol-L-arginine methylester (L-NAME) into cell cultures promoted cell expansion and reduced apoptosis. These results indicate that NO production originates from SC, but not from LNC. Low expression of co-stimulatory molecules and NOS2 of LNC limits NO induction. The high levels of NO derived from SC are involved in the self-limiting mechanisms of autoimmune responses by inhibiting cell expansion and promoting cell apoptosis.

An alternative pathway of nitric oxide production by rat astrocytes requires specific antigen and T cell contact.

In the present study, we observed an alternative pathway in which nitric oxide (NO) production by rat astrocytes requires specific antigen and cell-cell contact. NO production by astrocytes was significantly inhibited by antibodies against CD40L, B7-1 or B7-2. Astrocyte-derived NO inhibited T cell proliferation and induced T cell apoptosis. In contrast, augmented astrocyte proliferation was correlated to the levels of NO production by astrocytes, implicating a role of NO in regulating local immune responses in the central nervous system. These results suggest that T cell-astrocyte interactions may regulate local immune responses via the NO pathway and influence the fate of infiltrating T cells.