Y.-M. Huang

Multiple sclerosis is associated with an imbalance between tumour necrosis factor-alpha (TNF-alpha)- and IL-10-secreting blood cells that is corrected by interferon-beta (IFN-beta) treatment.

The up‐regulated B cell responses detectable in cerebrospinal fluid (CSF) and the augmented myelin antigen‐specific T cell responses observed in the CSF as well as systematically in patients with multiple sclerosis (MS) suggest the involvement of cytokines in disease development and perpetuation. Here we report on the parallel involvement of TNF‐α, IL‐6, IFN‐γ and IL‐10 in MS and controls, using enzyme‐linked immunospot (ELISPOT) assays to detect and enumerate cytokine‐secreting mononuclear cells (MNC) prepared from blood and, for IL‐6 and IL‐10, from CSF without in vitro stimulation. MS is associated with elevated levels of TNF‐α‐secreting blood MNC when compared with levels in groups of control patients with myasthenia gravis (MG) and other neurological diseases (OND) or healthy subjects. This elevation was confined to patients with untreated MS and not present in those examined during ongoing treatment with IFN‐β. Untreated patients with MS had lower numbers of IL‐10‐secreting blood MNC compared with the three control groups. In patients undergoing treatment with IFN‐β, numbers of IL‐10‐secreting cells were in the same range as in controls. Normalization of TNF‐α from elevated, and of IL‐10 from decreased levels could be one reason for the beneficial effects of IFN‐β in MS, although it remains to be shown whether these changes reflect phenomena primarily involved in MS pathogenesis or secondary changes. In CSF, levels of IL‐10‐secreting cells were higher than in blood in both MS and OND, with no difference between these groups. Systemic aberrations of IL‐6 and IFN‐γ and of IL‐6 in CSF in MS versus controls were only minor, irrespective of treatment with IFN‐β.

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.

Dendritic cells exposed in vitro to TGF-β1 ameliorate experimental autoimmune myasthenia gravis

Experimental autoimmune myasthenia gravis (EAMG) is an animal model for human myasthenia gravis (MG), characterized by an autoaggressive T‐cell‐dependent antibody‐mediated immune response directed against the acetylcholine receptor (AChR) of the neuromuscular junction. Dendritic cells (DC) are unique antigen‐presenting cells which control T‐ and B‐cell functions and induce immunity or tolerance. Here, we demonstrate that DC exposed to TGF‐β1 in vitro mediate protection against EAMG. Freshly prepared DC from spleen of healthy rats were exposed to TGF‐β1 in vitro for 48 h, and administered subcutaneously to Lewis rats (2 × 106DC/rat) on day 5 post immunization with AChR in Freund’s complete adjuvant. Control EAMG rats were injected in parallel with untreated DC (naive DC) or PBS. Lewis rats receiving TGF‐β1‐exposed DC developed very mild symptoms of EAMG without loss of body weight compared with control EAMG rats receiving naive DC or PBS. This effect of TGF‐β1‐exposed DC was associated with augmented spontaneous and AChR‐induced proliferation, IFN‐γ and NO production, and decreased levels of anti‐AChR antibody‐secreting cells. Autologous DC exposed in vitro to TGF‐β1 could represent a new opportunity for DC‐based immunotherapy of antibody‐mediated autoimmune diseases.

Elevated expression of CCR5 by myeloid (CD11c+) blood dendritic cells in multiple sclerosis and acute optic neuritis

Myeloid and plasmacytoid dendritic cells (DC) are present in cerebrospinal fluid (CSF) in non‐inflammatory neurological diseases (NIND) and elevated in clinically definite multiple sclerosis (MS) and in early MS – acute monosymptomatic optic neuritis (ON). Here, we show that expression of CCR5, a chemokine receptor for regulated on activation, normal T cell expressed and secreted (RANTES) and macrophage inflammatory protein (MIP)‐1α/β, is elevated on blood myeloid (CD11c+) DC in MS and ON compared to non‐inflammatory controls. In contrast, expression of CXCR4, a receptor for stromal cell‐derived factor (SDF)‐1α, is similar in all groups. Blood myeloid DC from MS patients respond chemotactically to RANTES and MIP‐1β, which are expessed in MS lesions. In active MS and ON, expression of CCR5 by myeloid DC in blood correlates with numbers of these cells in CSF. Thus, elevation of CCR5 may contribute to recruitment of myeloid DC to CSF in MS and ON. Recruitment of plasmacytoid DC to CSF appears to be CCR5‐independent.

Nasal tolerance in experimental autoimmune myasthenia gravis (EAMG): induction of protective tolerance in primed animals

Nasal administration of μg doses of acetylcholine receptor (AChR) is effective in preventing the development of B cell‐mediated EAMG in the Lewis rat, a model for human MG. In order to investigate whether nasal administration of AChR modulates ongoing EAMG, Lewis rats were treated nasally with AChR 2 weeks after immunization with AChR and Freunds complete adjuvant. Ten‐fold higher amounts of AChR given nasally (600 μg/rat) were required to ameliorate the manifestations of EAMG compared with the amounts necessary for prevention of EAMG. In lymph node cells from rats receiving 600 μg/rat of AChR, AChR‐induced proliferation and interferon‐gamma (IFN‐γ) secretion were reduced compared with control EAMG rats receiving PBS only. The anti‐AChR antibodies in rats treated nasally with 600 μg/rat of AChR had lower affinity, reduced proportion of IgG2b and reduced capacity to induce AChR degradation. Numbers of AChR‐reactive IFN‐γ and tumour necrosis factor‐alpha (TNF‐α) mRNA‐expressing lymph node cells from rats treated nasally with 600 μg/rat of AChR were suppressed, while IL‐4, IL‐10 and transforming growth factor‐beta (TGF‐β) mRNA‐expressing cells were not affected. Collectively, these data indicate that nasal administration of AChR in ongoing EAMG induced selective suppression of Th1 functions, i.e. IFN‐γ and IgG2b production, but no influence on Th2 cell functions. The impaired Th1 functions may result in the production of less myasthenic anti‐AChR antibodies and contribute to the amelioration of EAMG severity in rats treated with AChR 600 μg/rat by the nasal route.

Interferon-γ-modified dendritic cells suppress B cell function and ameliorate the development of experimental autoimmune myasthenia gravis

This study was designed to investigate the therapeutic effects of interferon (IFN)‐γ‐modulated dendritic cells (DC) in experimental autoimmune myasthenia gravis (EAMG). We induced EAMG in Lewis rats by immunization with Torpedo nicotinic acetylcholine receptor (nAChR) and adjuvant. On day 33 post‐immunization (p.i.), splenic DC were prepared, exposed to IFN‐γ alone (IFN‐γ‐DC) or to IFN‐γ in combination with 1‐methyl‐DL‐tryptophan (1‐MT), the specific inhibitor of indoleamine 2,3‐dioxygenase (IDO) (IFN‐γ + 1‐MT‐DC), and injected subcutaneously into rats with incipient EAMG on day 5 p.i. A control group of EAMG rats received naive DC on day 5 p.i., while another group received 1‐MT every other day, intraperitoneally (p.i.), from days 5 to 41 p.i. The severity of clinical signs of EAMG was reduced dramatically in IFN‐γ‐DC‐treated rats compared to rats receiving naive DC, IFN‐γ + 1‐MT‐DC or 1‐MT alone. The number of plasma cells secreting nAChR antibodies was reduced and the expression of B cell activation factor (BAFF) on splenic and lymph node mononuclear cells (MNC) was down‐regulated in rats treated with IFN‐γ‐DC. In vitro co‐culture of MNC derived from EAMG rats with IFN‐γ‐DC produced relatively few cells secreting nAChR antibodies. Addition of 1‐MT to the co‐culture significantly increased the number of cells secreting nAChR antibodies. We conclude that IFN‐γ‐DC reduced the number of plasma cells secreting nAChR antibodies in an IDO‐dependent manner and ameliorated the development of EAMG in Lewis rats.

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.

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.