Xue-Feng Bai

Nasal administration of myelin basic protein prevents relapsing experimental autoimmune encephalomyelitis in DA rats by activating regulatory cells expressing IL-4 and TGF-β mRNA

This study explores nasal administration of myelin basic protein (MBP) as a potential means of inducing tolerance to relapsing experimental autoimmune encephalomyelitis (PR-EAE), an experimental multiple sclerosis (MS) model that was induced in DA rats by immunization with rat spinal cord homogenate and incomplete Freunds adjuvant. DA rats received a total dosage of 0, 6, 60, 600 micrograms/rat of bovine MBP on ten consecutive days prior to immunization. EAE with typical course was observed in control rats receiving only PBS nasally, and in rats receiving 6 micrograms/rat of MBP. Rats receiving 60 micrograms/rat of MBP developed acute EAE but no relapse during 60 days of observation post immunization (p.i.). Only one of eight rats receiving 600 micrograms/rat of MBP developed slight, transient EAE. This protection was confirmed at the histology level and was associated with decreased levels of MBP-reactive IFN-gamma secreting Th1-like spleen cells on day 13 and 60 p.i. Rats receiving 60 and 600 micrograms/rat of MBP showed decreased serum anti-MBP IgG2b antibody levels on day 60 p.i., and rats receiving 600 micrograms/rat of MBP had marginally increased anti-MBP IgG1 antibody levels in serum compared to control EAE rats. Cytokine mRNA profiles in central nervous system (CNS) and spleen mononuclear cells were evaluated. Dose-dependent reduction of TNF-alpha mRNA expression were observed both in CNS and in splenocytes. Increased IL-4 and TGF-beta mRNA expression were observed in CNS of low (6 micrograms/rat) and median (60 micrograms/rat) dose of MBP tolerized rats and in splenocytes of rats tolerized with 600 micrograms/rat of MBP. We conclude that nasal administration of MBP in DA rat prevents EAE induced by immunization with whole rat spinal cord homogenate that, besides MBP, contains multiple antigenic myelin proteins. A mechanism involving MBP-reactive regulatory cells expressing IL-4 and TGF-beta mRNA acts as part in the induction of this tolerance.

Suppression of acute and protracted-relapsing experimental allergic encephalomyelitis by nasal administration of low-dose IL-10 in rats

In this study we report for the first time that nasal administration of the Th2 cell-related cytokine interleukin-10 (IL-10), at concentrations of 1.5 microg/rat and 15 microg/rat, suppressed clinical signs of acute experimental allergic encephalomyelitis (EAE) in Lewis rats and prevented the development and relapse of protracted-relapsing EAE (PR-EAE) in DA rats. In contrast, subcutaneous injection of IL-10 (15 microg/rat) did not inhibit acute EAE. The IL-10-mediated suppression of EAE was associated with decreased myelin antigen-specific T-cell proliferative responses and IFN-gamma secretion in both acute and PR-EAE. In sections of spinal cords derived from rats nasally pretreated with IL-10, there were no infiltrating CD4+ T cells or macrophages, which are considered as major encephalitogenic or inflammatory cells. Most interestingly, nasally administered IL-10 also inhibited MHC class II expression in microglia, indicating that IL-10 administration by the nasal route prevents the activation of microglia. Administration of cytokines via the nasal route offers an exciting alternative in the prevention and treatment of autoimmune diseases.

Transforming growth factor-β1 induces apoptosis of rat microglia without relation to bcl-2 oncoprotein expression

Transforming growth factor-beta (TGF-beta) plays a role as an immunosuppressive cytokine within the central nervous system (CNS). The CNS cells targeted by action of TGF-beta1 have not been defined. In this study, we tested the effect of TGF-beta1 on microglia, astrocytes and oligodendrocytes from newborn rats. TGF-beta1 selectively induced apoptosis of microglia, and not of astrocytes or oligodendrocytes. To study the apoptotic mechanism, bcl-2 oncoprotein expression in microglia, astrocytes and oligodendrocytes was measured. Bcl-2 was mainly expressed in microglia, indicating that microglial bcl-2 does not prevent TGF-beta1-mediated microglial apoptosis. The relative protein expression of bcl-2 in microglia was not related to frequency of microglial apoptosis. Thus, TGF-beta1-induced microglial apoptosis was regulated by a bcl-2-independent mechanism. Expression of cytokine (IL-1beta, IL-6, IL-12, IFN-gamma and TNF-alpha) mRNA on microglia was not influenced by treatment with TGF-beta1.

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.

Complexities of applying nasal tolerance induction as a therapy for ongoing relapsing experimental autoimmune encephalomyelitis (EAE) in DA rats

EAE is an autoimmune disease of the central nervous system (CNS) that serves as an experimental model for the human inflammatory demyelinating disease multiple sclerosis (MS). Antigen‐based immunotherapy including soluble antigen administration via feeding has been shown to be successful in treating EAE in rodents. In the present study, we explore nasal administration of small amounts of myelin basic protein (MBP) as a potential means of treatment of protracted, relapsing EAE (PR‐EAE) in a novel DA rat system. We found that nasal administration of MBP prevented EAE induced with whole spinal cord homogenate + Freunds incomplete adjuvant (FIA), and strongly down‐regulated levels of MBP‐reactive interferon‐gamma (IFN‐γ)‐secreting Th1‐like cells. However, in rats with ongoing PR‐EAE receiving the same regimen of MBP, a trend of aggravated disease was recorded, in conjunction with augmented levels of MBP‐reactive IFN‐γ‐secreting Th1‐like splenocytes during the acute phase of EAE. These data have implications for the clinical application of nasal tolerance to autoimmune diseases.

Linomide-induced suppression of experimental autoimmune neuritis is associated with down-regulated macrophage functions.

Experimental autoimmune neuritis (EAN) is a T-cell mediated autoimmune disease of the peripheral nervous system, in which macrophages and T-cells feature prominently in nerve lesions. EAN represents a counterpart to Guillain-Barré syndrome in humans. In the present study, we investigated the in vitro and in vivo effects of Linomide (LS-2616, quinoline-3-carboxamide), a synthetic immunomodulatory compound, on macrophages in relation to EAN. Linomide strongly suppressed IFN-gamma and lipopolysaccharide (LPS)-induced IL-1 beta, TNF-alpha and IL-6 mRNA expression in macrophages in vitro as demonstrated by in situ hybridisation. Linomide administered daily subcutaneously from the day of inoculation completely prevented the development of clinical symptoms of EAN. Linomide administered from day 9 post immunisation (p.i.) significantly suppressed clinical EAN. Macrophages from Linomide-treated EAN rats showed decreased IL-1 beta, TNF-alpha and IL-6 mRNA expression in response to IFN-gamma and LPS. LPS-induced nitric oxide production by macrophages was also suppressed by Linomide in vitro. Linomide, however, does not affect macrophage death and release of lactate dehydrogenase. We conclude that Linomide may exert its actions in EAN and perhaps also in other autoimmune disease models, by suppressing macrophage functions.

Nasal administration of multiple antigens suppresses experimental autoimmune myasthenia gravis, encephalomyelitis and neuritis

Oral tolerization with acetylcholine receptor (AChR) and myelin basic protein (MBP) prior to immunization with AChR+MBP+ complete Freunds adjuvant (CFA) alleviated clinical signs of experimental autoimmune myasthenia gravis (EAMG)+experimental allergic encephalomyelitis (EAE) and AChR- or MBP-specific T and B cell responses. Tolerance induced via the nasal route needs much less tolerogen and may still be as effective as oral tolerance induction. We now immunized Lewis rats with AChR+MBP+bovine peripheral nerve myelin (BPM)+CFA, which resulted in a multiphasic clinical picture with a combination of clinical signs of the EAMG+EAE+experimental allergic neuritis (EAN), accompanied by massive macrophage infiltrations in sections of muscle, spinal cord and sciatic nerve, and strong T and B cell responses to AChR, MBP and BPM in lymphoid organs. Nasal administration of microg doses of AChR+MBP+BPM prior to immunization with a mixture of these antigens+CFA effectively suppressed the incidence and severity of clinical disease, reduced macrophage infiltrations in sections of muscle, spinal cord and sciatic nerve, and down-regulated autoreactive T cell responses to the three antigens in lymphoid organs. Numbers of AChR-, MBP-, BPM-reactive Th1 type of cytokine interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha mRNA expression in lymph node cells were markedly suppressed, while transforming growth factor-beta (TGF-beta) mRNA expression was upregulated from nasally tolerized rats, suggesting an active suppression mechanism may act partly in the induction of tolerance. The results implicate the possibility to establish multiple autoantigen-based vaccination for the prevention of autoimmune diseases in humans.

Dose-dependent mechanisms relate to nasal tolerance induction and protection against experimental autoimmune encephalomyelitis in Lewis rats.

Nasal administration of soluble antigens is an exciting means of specifically down‐regulating pathogenic T‐cell reactivities in autoimmune diseases. The mechanisms by which nasal administration of soluble antigens suppresses autoimmunity are poorly understood. To define further the principles of nasal tolerance induction, we studied the effects of nasal administration of myelin basic protein (MBP) on experimental autoimmune encephalomyelitis (EAE) in the Lewis rat. EAE is a CD4+ T‐cell‐mediated animal model for human multiple sclerosis. Nasal administration of guinea‐pig (gp)‐MBP at a dose as low as 30 μg/rat can completely prevent gp‐MBP‐induced EAE, whereas nasal administration of bovine (b)‐MBP is not effective even at a much higher dosage. Cellular immune responses, as reflected by T‐cell proliferation and interferon‐γ (IFN‐γ)‐ELISPOT, were suppressed in rats receiving the two different doses (30 and 600 μg/rat) of gp‐MBP, but not after administration of b‐MBP. Rats tolerized with both doses of gp‐MBP had also abrogated MBP‐induced IFN‐γ mRNA expression in popliteal and inguinal lymph node mononuclear cells compared with rats receiving phosphate‐buffered saline nasally. However, adoptive transfer revealed that only spleen mononuclear cells from rats pretreated with a low dose, but not from those pretreated with a high dose, of gp‐MBP transferred protection to actively induced EAE. Low‐dose (30 μg/rat) gp‐MBP‐tolerized rats also had high numbers of interleukin‐4 (IL‐4) mRNA‐expressing lymph node cells, while high‐dose (600 μg/rat) gp‐MBP‐tolerized rats had low numbers of IL‐4 mRNA‐expressing lymph node cells. Our data suggest an exquisite specificity of nasal tolerance. Dose‐dependent mechanisms also relate to nasal tolerance induction and protection against EAE in the Lewis rat.

Experimental allergic neuritis: cytolysin mRNA expression is upregulated in lymph node cells during convalescence

Experimental allergic neuritis (EAN) is a T cell mediated animal model of Guillain-Barré syndrome, characterized by inflammation and demyelination of the peripheral nervous system (PNS). To study the involvement of immunoregulatory cytokines, we induced EAN in Lewis rats by immunizing with bovine PNS myelin (BPM) and Freunds complete adjuvant. mRNA expression of the cytokines IL-1beta, IL-6, IL-10, IL-12, TNF-alpha and TNF-beta, and the cytolytic effector molecule cytolysin was examined in lymph node mononuclear cells (MNC) over the course of EAN by in situ hybridization after culture without antigen and in the presence of BPM, the myelin P2 protein, the control antigen acetylcholine receptor, or the mitogen PHA. Three patterns of cytokine mRNA expressing MNC in relation to clinical EAN could be distinguished: (i) IL-1beta mRNA expressing cells peaked already on day 3 post immunization (p.i.), and BPM- and P2-reactive TNF-alpha, and BPM-reactive IL-6 mRNA expressing cells were also detected already on day 7 p.i., i.e., before onset of clinical EAN; (ii) BPM- and P2-reactive TNF-alpha peaked together with P2-reactive TNF-beta, IL-6 and IL-12 mRNA expressing cells at height of clinical EAN, consistent with a disease-promoting role for these four cytokines; (iii) high levels of BPM- and P2-reactive IL-10 and cytolysin mRNA expressing cells were observed only during recovery (day 28 p.i.), consistent with a disease down-regulating role of IL-10 and cytolysin. The results suggest a major proinflammatory role for IL-1beta, TNF-alpha, TNF-beta, IL-6 and IL-12 and a disease down-regulating function of IL-10 as well as cytolysin in EAN.

Cellular mRNA expression of interferon‐gamma (IFN‐γ), IL‐4 and IL‐10 relates to resistance to experimental autoimmune myasthenia gravis (EAMG) in young Lewis rats

Age‐related alterations in the immune system, including changes in lymphocyte subset composition, result in changes of cytokine patterns and might thereby influence the incidence and severity of autoimmune diseases. To investigate the age‐related resistance to EAMG, an animal model for human MG, young (4‐week‐old) and adult (8–10‐week‐old) female Lewis rats were immunized with Torpedo acetylcholine receptor (AChR) and Freunds complete adjuvant (FCA). Adult Lewis rats showed severe weight loss and progressive muscular weakness after immunization, while young rats developed minor clinical signs of EAMG after a prolonged interval post‐immunization. By comparison with adult rats, the young had lower AChR‐specific T and B cells responses, and less muscle AChR loss. In situ hybridization performed on mononuclear cells (MNC) from lymph nodes revealed that young rats had lower levels of AChR‐specific IFN‐γ, IL‐4 and IL‐10 mRNA‐expressing cells compared with adult rats. Since IFN‐γ, IL‐4 and IL‐10 promote the development of EAMG, the low expression of these cytokines might contribute to EAMG resistance in young Lewis rats.