Hulun Li

Natural killer cells determine the outcome of B cell-mediated autoimmunity.

Natural killer (NK) cells can affect the outcome of adaptive immune responses. NK cells, but not NK1.1+ T cells, were found to participate in the development of myasthenia gravis (a T cell–dependent, B cell– and antibody-mediated autoimmune disease) in C57BL/6 mice. The requirement for NK cells was reflected by the lack of a type 1 helper T cell response and antibodies to the acetylcholine receptor in both NK1.1+ cell–depleted and NK cell–deficient IL-18−/− mice. These findings establish a previously unrecognized link between NK cells and autoreactive T and B cells.

IL-17 and IFN-γ mRNA expression is increased in the brain and systemically after permanent middle cerebral artery occlusion in the rat

Brain ischemia is characterized by local inflammation reflected by accumulation of inflammatory cells and a multitude of mediators. Among them, cytokines and chemokines may influence the inflammatory cascade that follows cerebral ischemia. Here we report on brain hemispheric and systemic increase of pro-inflammatory IL-17 and IFN-gamma, the anti-inflammatory cytokines IL-4 and IL-10, and the chemokines IP-10, IL-8 and MIP-2, 1 h to 6 days after permanent middle cerebral artery occlusion (pMCAO). IL-17 and IFN-gamma mRNA levels were elevated in the ischemic hemispheres of pMCAO-operated rats compared with corresponding hemispheres of sham-operated rats. Levels were slightly elevated at 1 h, and peaked at 6 days after pMCAO. IL-8 and MIP-2 levels in the ischemic hemispheres peaked at 24 h, whereas IP-10 showed a biphasic profile with two peaks at 6 h and 6 days after pMCAO. IL-4 peaked in the ischemic hemispheres at 6 h, when IL-10 levels were lower than in sham-operated rats, and IL-10 levels peaked at 2 days after pMCAO. Systemically, the numbers of IL-17 and IFN-gamma mRNA expressing blood mononuclear cells were elevated already at 1 h after pMCAO, preceding the changes in the ischemic hemispheres. Altered levels of IL-17 and IFN-gamma after pMCAO may affect outcome of brain ischemia.

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.

Anti-CTLA-4 Antibody Treatment Triggers Determinant Spreading and Enhances Murine Myasthenia Gravis

CTLA-4 appears to be a negative regulator of T cell activation and is implicated in T cell-mediated autoimmune diseases. Experimental autoimmune myasthenia gravis (EAMG), induced by immunization of C57BL/6 mice with acetylcholine receptor (AChR) in adjuvant, is an autoantibody-mediated disease model for human myasthenia gravis (MG). The production of anti-AChR Abs in MG and EAMG is T cell dependent. In the present study, we demonstrate that anti-CTLA-4 Ab treatment enhances T cell responses to AChR, increases anti-AChR Ab production, and provokes a rapid onset and severe EAMG. To address possible mechanisms underlying the enhanced autoreactive T cell responses after anti-CTLA-4 Ab treatment, mice were immunized with the immunodominant peptide α146–162 representing an extracellular sequence of the AChR. Anti-CTLA-4 Ab, but not control Ab, treatment subsequent to peptide immunization results in clinical EAMG with diversification of the autoantibody repertoire as well as enhanced T cell proliferation against not only the immunizing α146–162 peptide, but also against other subdominant epitopes. Thus, treatment with anti-CTLA-4 Ab appears to induce determinant spreading, diversify the autoantibody repertoire, and enhance B cell-mediated autoimmune disease in this murine model of MG.

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.

Suppression of autoimmune neuritis in IFN-γ receptor-deficient mice

1 T cells mediate demyelination in the peripheral nervous system (PNS). Infiltrating macrophages and T cells as well as cytokines like interferon (IFN)-g are intimately involved in causing pathogenic effects. To investigate the role of IFN-g in cell-mediated EAN, IFN-g receptor-deficient mutant (IFN-gR 2/2 ) C57BL/6 mice and corresponding wild-type mice were immu- nized with P0 peptide 180 -199, a purified component of peripheral nerve myelin, and Freunds complete adjuvant. IFN-gR 2/2 mice exhibited later onset of clin- ical disease. The disease was also less severe than in wild-type mice. Fewer IL-12-producing but more IL-4- producing cells were found in sciatic nerve sections from IFN-gR

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.

CD28–B7 costimulation: a critical role for initiation and development of experimental autoimmune neuritis in C57BL/6 mice

CD28 provides a critical costimulatory signal for antigen-specific T cell activation. Because CD28 is an important factor in the development of autoimmune diseases, we investigated its role in T cell-mediated experimental autoimmune neuritis (EAN), an animal model of Guillain-Barré syndrome in humans. CD28-deficient mutant (CD28-/-) C57BL/6 mice and corresponding wild-type mice were immunized with P0 peptide 180-199, a purified component of peripheral nerve myelin, and Freunds complete adjuvant. As a result, all wild-type mice developed severe EAN, in contrast, none of the CD28-/- mice manifested clinical signs of disease. Additionally, CD28-/- mice had fewer IL-12 producing cells in sciatic nerve sections and fewer IFN-gamma secreting splenic cells than wild-type mice on day 24 post immunization, i.e., at the peak of clinical EAN. At that time point, CD28-/- mice had milder infiltration of such inflammatory cells as macrophages, CD4+ T cells and monocytes into sciatic nerve tissues and less demyelination than wild-type mice. Moreover, the CD28-deficiency led to reduced production of specific anti-P0 peptide 180-199 antibodies compared with wild-type mice. Evidently, CD28 is required for interaction with B7 to regulate the activation of T and B cells that initiates development of EAN.

Cytokine and chemokine mRNA expressing cells in muscle tissues of experimental autoimmune myasthenia gravis

In-situ hybridization with labeled oligonucleotide probes was applied to explore cytokine and chemokine mRNA expression in sections of striated muscle, the target organ in experimental autoimmune myasthenia gravis (EAMG), induced in Lewis rats by immunization with acetylcholine receptor (AChR) and complete Freunds adjuvant (CFA). A transient burst of TNF-alpha, IL-1beta and IL-6 mRNA expressing cells was detected during the early phase of EAMG. This cytokine pattern was related to muscular infiltration of macrophages. Levels of IL-4, IL-10, IFN-gamma, cytolysin and TGF-beta mRNA expressing cells were low and observed mainly during the early phase of EAMG. C-C chemokine RANTES, MCP, MIP-1alpha and MIP-2 mRNA expressing cells were not detected over the course of EAMG. The low and transient expression of cytokines in EAMG muscle tissues suggests that the immune effector responses are unlikely operated by infiltrating cells in muscle. Muscular infiltrations in EAMG are unlikely due to local accumulation of C-C chemokines.