Inhibition of tyrosine kinase signaling by tyrphostin AG126 downregulates the IL-21/IL-21R and JAK/STAT pathway in the BTBR mouse model of autism

Autism spectrum disorder (ASD) comprises a broad range of neurodevelopmental disorders that are associated with deficits in social interaction and communication. The tyrosine kinase inhibitor tyrphostin AG126 represents a promising therapeutic agent for several neuroinflammatory disorders. There are currently no treatments available that can improve ASD and we previously showed that AG126 treatment exerts beneficial effects on BTBR T⁺ Itpr3^tf/J (BTBR) mice, a model for autism that shows the core features of ASD; however, the immunological mechanisms and molecular targets associated with this effect were previously unclear. This study was undertaken to delineate the neuroprotective effect of AG126 on BTBR mice. Here, using this mouse model, we investigated the effects of AG126 administration on IL-21R, IL-21, IL-22, TNF-α, NOS2, STAT3, IL-27, and Foxp3 production by CD8⁺ T cells in the spleen by flow cytometry. We further explored the mRNA and protein expression of IL-21, IL-22, IL-1β, TNF-α, NOS2, JAK1, STAT3, IL-27, and Foxp3 in brain tissue by RT-PCR, and western blotting. We found that BTBR mice treated with AG126 exhibited significant decreases in IL-21R-, IL-21-, IL-22-, TNF-α-, NOS2-, STAT3-producing, and increases in IL-27- and Foxp3-producing, CD8⁺ T cells. Our results further demonstrated that AG126 treatment effectively decreased IL-21, IL-22, IL-1β, TNF-α, NOS2, JAK1, and STAT3, and increased IL-27 and Foxp3 mRNA and protein expression in brain tissues. Our findings suggest that AG126 elicits a neuroprotective response through downregulation of the IL-21/IL-21R and JAK/STAT pathway in BTBR mice, which could represent a promising novel therapeutic target for ASD treatment.     

Methylmercury chloride exposure aggravates proinflammatory mediators and Notch-1 signaling in CD14+ and CD40+ cells and is associated with imbalance of neuroimmune function in BTBR T+ Itpr3tf/J mice

Autism spectrum disorder (ASD) is a severe neurodevelopmental disorder characterized by deficits in social interaction, communication, and repetitive behaviors. A key role for immune dysfunction has been suggested in ASD. Recent studies have indicated that inflammatory mediators and Notch-1 signaling may contribute to the development of ASD. Methylmercury chloride (MeHgCl) is an environmental pollutant that primarily affects the central nervous system, causing neurological alterations. Its effects on immunological responses have not been fully investigated in ASD. In this study, we examined the influence of MeHgCl exposure on inflammatory mediators and Notch-1 signaling in BTBR T⁺ Itpr3tf/J (BTBR) mice, a model of ASD. We examined the effects of MeHgCl on the IL-6-, GM-CSF-, NF-κB p65-, Notch-1-, and IL-27-producing CD14⁺ and CD40⁺ cells in the spleen. We assessed the effect of MeHgCl on IL-6, GM-CSF, NF-κB p65, Notch-1, and IL-27 mRNA levels in brain tissue. We also measured IL-6, GM-CSF, and NF-κB p65 protein expression levels in brain tissue. MeHgCl exposure of BTBR mice significantly increased IL-6-, GM-CSF-, NF-κB p65-, and Notch-1-, and decreased IL-27-producing CD14⁺, and CD40⁺ cells in the spleen. MeHgCl exposure of BTBR mice upregulated IL-6, GM-CSF, NF-κB p65, and Notch-1, and decreased IL-27 mRNA expression levels in brain tissue. Moreover, MeHgCl resulted in elevated expression of the IL-6, GM-CSF, and NF-κB p65 proteins in brain tissue. Taken together, these results indicate that MeHgCl exposure aggravates proinflammatory mediators and Notch-1 signaling which are associated with imbalance of neuroimmune function in BTBR mice.     

Autism spectrum disorders may be due to cerebral toxoplasmosis associated with chronic neuroinflammation causing persistent hypercytokinemia that resulted in an increased lipid peroxidation,oxidative stress,and depressed metabolism of endogenous and exogenous substances

Worldwide, approximately 2 billion people are chronically infected with Toxoplasma gondii with largely yet unknown consequences. Patients with autism spectrum disorders (ASD) similarly as mice with chronic toxoplasmosis have persistent neuroinflammation, hypercytokinemia with hypermetabolism associated with enhanced lipid peroxidation, and extreme changes in the weight resulting in obesity or wasting. Data presented in this review suggest that environmental triggering factors such as pregnancy, viral/bacterial infections, vaccinations, medications, and other substances caused reactivation of latent cerebral toxoplasmosis because of changes in intensity of latent central nervous system T. gondii infection/inflammation and finally resulted in development of ASD. Examples of such environmental factors together with their respective biomarker abnormalities are: pregnancy (increased NO, IL-1β, TNF-α, IL-6, IL-10, prolactin; decreased IFN-γ, IL-12), neuroborreliosis (increased IL-1β, sIL-1R2, TNF-α, IFN-γ, IL-6, IL-10, IL-12, IL-18, transforming growth factor-β1 (TGF-β1)), viral infections (increased IL-1β, IL-6, IL-8, TNF-α, IFN-γ/α/β, TGF-β1), thimerosal (increased IL-5, IL-13; decreased IFN-γ, TNF-α, IL-6, IL-12p70, NOS), and valproic acid (increased NO, reactive oxygen species; decreased TNF-α, IL-6, IFN-γ). The imbalances in pro- and antiinflammatory processes could markedly hinder host defense mechanisms important for immune control of the parasite, such as the production of NO, cytokines, and reactive oxygen/nitrogen species, tryptophan degradation by indoleamine 2,3-dioxygenase and/or tryptophan 2,3-dioxygenase, limitation of the availability of intracellular iron to T. gondii, and the mechanisms mediated by an IFN-γ responsive gene family. These fluctuations could result in a recurrent profuse multiplication of T. gondii in the brain associated with persistent neuroinflammation, chronic overproduction of pro- and antiinflammatory cytokines, and NO causing increased oxidative stress, and significantly depressed activity of several enzymes including cytochrome P450 monooxygenase family responsible for metabolism of physiological substrates and xenobiotics, such as steroids, fatty acids, prostaglandins, drugs, pollutants, and carcinogens, finally leading to development of ASD. This reasoning may be supported by such abnormal metabolic events as: (1) patients with ASD have significantly decreased melatonin levels caused by marked deficit in acetylserotonin methyltransferase activity, possibly resulting from maternal and/or fetal/postnatal overproduction of NO, characteristic for this clinical entity; (2) thimerosal inhibited both insulin-like growth factor-1- and dopamine-stimulated methylation reactions, and depressed methionine synthase activity, the metabolic events important for promoting normal neurodevelopment; (3) valproic acid, a strong histone deacetylase inhibitor, have potent anti-T. gondii activity. Thus, patients with ASD should be tested for T. gondii infection.     

Resveratrol treatment attenuates chemokine receptor expression in the BTBR T + tf/J mouse model of autism

Autism is a neurodevelopmental disorder categorized by qualitative impairments in social interaction, communication, and repetitive stereotypic behavior. Emerging evidence increasingly suggests that chemokine receptors have a pivotal role in the central nervous system and are involved in the pathogenesis of numerous neuroinflammatory diseases. Resveratrol is widely used to treat neurodegenerative diseases, but its effect on autism has not been investigated. We investigated the effect of resveratrol (20 and 40 mg/kg) in the spleen and brain tissues of BTBR T + tf/J (BTBR) and C57BL/6J (B6) mice as well as on the C-C chemokine receptor (CCR) and C-X-C motif chemokine receptor (CXCR) (CCR3⁺, CCR5⁺, CCR7⁺ and CCR9⁺, CXCR3⁺ and CXCR5⁺) in cluster of differentiation 4-positive (CD4⁺) T cells in the spleen. We also assessed the mRNA expression of CCR and CXCR receptors in the spleen and brain tissues. Our study revealed that the BTBR and B6 control mice showed different immune profiles. The BTBR mice showed characteristic higher levels of both CCR and CXCR production and expression in CD4⁺ T cells than the B6 control mice did. Treatment of B6 and BTBR mice with resveratrol (20 and 40 mg/kg) induced a substantial decrease in the CCR and CXCR production and expression in CD4⁺ T cells compared with the respective untreated control groups. Moreover, resveratrol treatment decreased the mRNA expression levels of CCR and CXCR in the spleen and brain tissues. Resveratrol downregulated the chemokine receptor levels, which might provide unique targets for future therapies for autism.     

Infiltration of Th1 and Th17 cells and activation of microglia in the CNS during the course of experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is a mouse model for multiple sclerosis, where disease is mediated by autoantigen-specific T cells. Although there is evidence linking CD4⁺ T cells that secrete IL-17, termed Th17 cells, and IFN-γ-secreting Th1 cells with the pathogenesis of EAE, the precise contribution of these T cell subtypes or their associated cytokines is still unclear. We have investigated the infiltration of CD4⁺ T cells that secrete IFN-γ, IL-17 or both cytokines into CNS during development of EAE and have examined the role of T cells in microglial activation. Our findings demonstrate that Th17 cells and CD4⁺ T cells that produce both IFN-γ and IL-17, which we have called Th1/Th17 cells, infiltrate the brain prior to the development of clinical symptoms of EAE and that this coincides with activation of CD11b⁺ microglia and local production of IL-1β, TNF-α and IL-6 in the CNS. In contrast, significant infiltration of Th1 cells was only detected after the development of clinical disease. Co-culture experiments, using mixed glia and MOG-specific T cells, revealed that T cells that secreted IFN-γ and IL-17 were potent activators of pro-inflammatory cytokines but T cells that secrete IFN-γ, but not IL-17, were less effective. In contrast both Th1 and Th1/Th17 cells enhanced MHC-class II and co-stimulatory molecule expression on microglia. Our findings suggest that T cells which secrete IL-17 or IL-17 and IFN-γ infiltrate the CNS prior to the onset of clinical symptoms of EAE, where they may mediate CNS inflammation, in part, through microglial activation.     

Neuronal α1/2-adrenergic stimulation of IFN-γ, IL-6, and CXCL-1 in murine spleen in late experimental arthritis

Objective: Functional cross-talk exists between sympathetic nerve fibers and cytokine-producing splenic cells in early collagen type II–induced arthritis (CIA) (day 32). These earlier experiments demonstrated exclusively neuronal sympathetic regulation of IFN-γ, CXCL1, IL-6, and TGF-β. However, in late arthritis, the sympathetic influence might change due to loss of sympathetic nerve fibers and appearance of neurotransmitter-producing cells. We aimed to investigate neurotransmitter-dependent regulation of IFN-γ, CXCL1, IL-6, and TGF-β in murine spleen in late CIA.Methods: Spleens were removed when animals reached day 58 (46–68) after immunization to generate 0.35 mm-thick spleen slices, which were transferred to superfusion microchambers to electrically induce release of neurotransmitters. Using respective neurotransmitter antagonists, effects of released neurotransmitters on cytokine secretion were investigated.Results: There was electrically induced inhibition of IFN-γ, CXCL1, and IL-6, and stimulation of TGF-β, which was much less pronounced than in early CIA. There existed β adrenergic inhibition of IFN-γ, IL-6, and TGF-β (and stimulation of CXCL1) independent of electrical stimulation (interpreted as non-neuronal). However, there was a neuronal α1/2 adrenergic stimulation of IFN-γ, CXCL1, and IL-6 and, we observed neuronal A1-adenosinergic stimulation of TGF-β.Conclusions: In the late phase of CIA, non-neuronal modulation of cytokine secretion increases while neuronal regulation strikingly decreases. Particularly, β-adrenergic effects are non-neuronal while α1/2-adrenergic effects are clearly neuronal. We suggest that alterations in sympathetic innervation of the spleen fundamentally change the functional neuroimmune interplay in the spleen of arthritic mice.     

Respiratory infection with a bacterial pathogen attenuates CNS autoimmunity through IL-10 induction

Infection with viral or bacterial pathogens has been linked with the development of multiple sclerosis (MS), while infection with helminth parasites has been associated protection against MS and other autoimmune diseases. Here we have used a murine model of MS, experimental autoimmune encephalomyelitis (EAE), to examine the effect of infection with the respiratory pathogen Bordetella pertussis infection on development of CNS inflammation. The data demonstrate that infection of mice with B. pertussis significantly attenuates the clinical course of EAE induced by active immunization or cell transfer. This was reflected in a significant reduction in VLA-4 and LFA-1 expression on T cells and infiltration of IL-17⁺, IFN-γ⁺ and IFN-γ⁺IL-17⁺ CD4 T cells into the CNS. Infection with B. pertussis induced IL-10 production from dendritic cells in vitro and enhanced the frequency of IL-10-producing CD25^-Foxp3^+/− CD4⁺ T cells in vivo. Furthermore, the suppressive effects of B. pertussis infection on EAE were lost in IL-10^−/− mice. Our findings demonstrate that a bacterial infection of the respiratory tract can attenuate EAE by promoting production of the anti-inflammatory cytokine IL-10 that may suppress licensing of autoaggressive T cells in the lungs, thereby preventing their migration into the CNS.     

Transforming growth factor-β1 suppresses autoantigen-induced expression of pro-inflammatory cytokines but not of interleukin-10 in multiple sclerosis and myasthenia gravis

Multiple sclerosis (MS) is associated with high levels of circulating T lymphocytes that respond to the myelin antigens myelin basic protein (MBP) and proteolipid protein (PLP) by producing various cytokines including interferon-γ (IFN-γ) that makes MS worse and transforming growth factor-β (TGF-β), an endogenously produced immunosuppressant that might act beneficially. To further define the role of TGF-β in MS, we examined the effects of recombinant TGF-β1 (rTGF-β1) on autoantigen-mediated regulation of cytokines in MS and myasthenia gravis (MG). Blood mononuclear cells (MNC) were cultivated with or without rTGF-β1, and with or without autoantigen or the recall antigen PPD. MNC expressing cytokine mRNA were detected after in situ hybridization with radiolabeled cDNA oligonucleotide probes. Femtogram concentrations of rTGF-β1 suppressed MBP-, PLP- and PPD-induced upregulation of IFN-γ, IL-4, IL-6, tumor necrosis factor-α (TNF-α), TNF-α and perforin in MS, and acetylcholine receptor (AChR)-induced augmentation of these pro-inflammatory cytokines in MG, but had no effects on autoantigen- or PPD-induced expression of IL-10 or TGF-β itself. rTGF-β1 also suppressed numbers of myelin antigen-reactive IFN-γ- and IL-4-secreting cells in MS and AChR-reactive IFN-γ and IL-4 secreting cells in MG. The selective suppressive effects of TGF-β1 on autoantigen-induced upregulation of pro-inflammatory cytokines makes TGF-β1 attractive as a treatment alternative in MS and MG.     

Sympathetic inhibition of IL-6, IFN-γ, and KC/CXCL1 and sympathetic stimulation of TGF-β in spleen of early arthritic mice

Objectives

The connection between sympathetic nerve fibers and immune cells in the spleen is known. In the context of arthritis, the functional meaning of the neuroimmune contact remains unclear. From immunization until disease outbreak, the sympathetic nervous system (SNS) has a proinflammatory influence which is converted into an anti-inflammatory influence after disease outbreak. This study investigated the influence of neuronally released neurotransmitters on IFN-γ, KC (CXCL1), IL-6, and TGF-β in spleen of mice shortly after outbreak of collagen type II-induced arthritis.

Methods

Spleens were removed when animals reached an arthritis score of 3 on a scale of 1–16 (approx. on day 32) in order to generate 0.35 mm-thick spleen slices. Spleen slices were transferred to superfusion microchambers in order to electrically induce release of sympathetic neurotransmitters. By means of this technique, the effect of physiologically released neurotransmitters was investigated on secretion of IFN-γ, KC, IL-6, and TGF-β.

Results

High amounts of IFN-γ, KC, IL-6, and TGF-β were released from superfused spleen, and electrical stimulation markedly inhibited IFN-γ, KC, and IL-6 release but pronouncedly stimulated TGF-β. The adrenergic influence via β-adrenoceptors stimulated release of IL-6 and, particularly, TGF-β. However, catecholamines inhibit release of IL-6 via α1-adrenergic pathways but without any effect on TGF-β. The co-transmitter adenosine stimulated IL-6 release via A1-adenosine receptors but no influence was recognized on TGF-β.

Conclusion

At disease outbreak, electrically released endogenous neurotransmitters of the SNS inhibit IFN-γ, KC, and IL-6 but β-adrenergically stimulate TGF-β. This creates an anti-inflammatory milieu that might be responsible for the observed dual influence of the SNS on arthritis.     

Multiple sclerosis: myelin basic protein induced mRNA expression of proinflammatory cytokines in mononuclear cells is suppressed by interferon-β 1b in vitro

Beta-interferon (IFN-β) is a promising treatment in multiple sclerosis (MS), reducing the exacerbation rate and MRI lesion burden, as well as the disease progression in relapsing-remitting MS. IFN-β was originally defined by its antiviral effects, but the interest has recently been focused on its immunomodulatory properties. Myelin basic protein (MBP) is one of several autoantigens considered to be the target for autoaggressive immune responses, which eventually might lead to the development of MS. To study in-vitro effects of IFN-β1b on MBP induced cytokine expression, mRNA for the Th1 cytokines IFN-γ and TNF-α, the Th2 related IL-4 and IL-6, the cytolytic perforin and the immune response downregulating TGF-β was measured with in situ hybridization after culture of blood mononuclear cells (MNC) in the presence and absence of MBP. Numbers of cells expressing IFN-γ, TNF-α, perforin and IL-4 mRNA were significantly suppressed after culture with 10 U/ml IFN-β1b. No such effect was seen on MBP induced IL-6 or TGF-β mRNA expression. These observations suggest that one of the major effects of IFN-β1b is the induction of a shift in the cytokine mRNA profile towards a more immunosuppressive pattern. In parallel in vitro tests, the control substance dexametasone (40 μg/ml) reduced the numbers of cells expressing mRNA for all cytokines under study with the exception of TGF-β, to an extent equal to or even more pronounced than IFN-β1b.     

Cytokines and the pathogenesis of myasthenia gravis

Myasthenia gravis (MG) and its animal model experimental autoimmune myasthenia gravis (EAMG) are caused by autoantibodies against nicotinic acetylcholine receptor (AChR) in skeletal muscle. The production of anti-AChR antibodies is mediated by cytokines produced by CD4⁺ and CD8⁺ T helper (Th) cells. Emerging investigations of the roles of cytokines in MG and EAMG have revealed that the Th2 cell related cytokine interleukin 4 (IL-4), an efficient growth promoter for B-cell proliferation and differentiation, is important for anti-AChR antibody production. IL-6 and IL-10 have similar effects. The Th1 cytokine IFN-γ is important in inducing B-cell maturation and in helping anti-AChR antibody production and, thereby, for induction of clinical signs and symptoms. Results from studies of time kinetics of cytokines imply that IFN-γ is more agile at the onset of EAMG, probably being one of the initiating factors in the induction of the disease, and IL-4 may be mainly responsible for disease progression and persistance. Even though other Th1 cytokines like IL-2, tumor necrosis factor α (TNF-α), and TNF-β as well as the cytolytic compound perforin do not directly play a role in T-cell-mediated help for anti-AChR antibody production, they are actually involved in the development of both EAMG and MG, probably by acting in concert with other cytokines within the cytokine network. In contrast, transforming growth factor β (TGF-β) exerts immunosuppressive effects which include the down-regulation of both Th1 and Th2 cytokines in MG as well as EAMG. Suppressive effects are also exerted by interferon α (IFN-α). Based on elucidation of the role of cytokines in EAMG and MG, treatments that up-modulate TGF-β or IFN-α and/or suppress cytokines that help B-cell proliferation could be useful to improve the clinical outcome. © 1997 John Wiley & Sons, Inc. Muscle Nerve, 20, 543–551, 1997     

Therapeutic gene silencing with siRNA for IL-23 but not for IL-17 suppresses the development of experimental autoimmune encephalomyelitis in rats

Gene silencing with siRNAs is important as a therapeutic tool in autoimmune diseases. In this study, we administered siRNAs specific for cytokines that may be involved in pathogenesis of experimental autoimmune encephalomyelitis (EAE). siRNA specific for IL-23p19 (siRNA-IL-23) suppressed EAE almost completely, whereas siRNA-IL-17A did not modulate the clinical course. Flow cytometric analysis revealed that siRNA-IL-23 significantly reduced the proportion of both IFN-γ⁺IL-17^? Th1 and IFN-γ^?IL-17⁺ Th17 cells in the spinal cord. Consistent with this finding, siRNA-IL-23 treatment downregulated IL-12, IL-17 and IL-23 mRNAs. These findings indicate that IL-23, but not IL-17, play an important role in the development of EAE.     

TGF-β1 affects endothelial cell interaction with macrophages and T cells leading to the development of cerebrovascular amyloidosis

Astrocyte-endothelial cell (EC) interactions play a major role in the function of the neurovascular unit. Dysfunction in these interactions may lead to amyloid accumulation in blood vessels and may cause microhemorrhage and cognitive impairment. Transforming growth factor-β1 (TGF-β1) expression levels positively correlate with the degree of cerebrovascular amyloid in Alzheimer’s disease (AD) cases. Furthermore, expression of TGF-β1 driven by the GFAP promoter in mice leads to an age-related deposition of amyloid, such as β-amyloid (Aβ), around cerebral blood vessels. Here, we demonstrate that TGF-β1 affects the cross talk between EC and inflammation, leading to a reduction in macrophage activity as measured by protein levels and migration ability. Changes in EC secreted factors following TGF-β1 stimulation also affect CD4⁺ T cell activation, as shown by a reduction in the levels of IFN-γ. Moreover, while medium from EC can stimulate macrophages to clear insoluble cerebrovascular amyloid from an AD mouse brain, pre-incubation of EC with TGF-β1 reduces the ability of EC to affect macrophage activity. Our findings support the importance of cross talk between EC, macrophages and CD4⁺ T cells in preventing cerebrovascular amyloid deposition. Understanding EC-immune system interactions may pave the way to new therapeutic approaches for cerebrovascular amyloidosis diseases.     

Genetic control of multiple sclerosis: Increased production of lymphotoxin and tumor necrosis factor-α by HLA-DR2+ T cells

Lymphotoxin (LT) and tumor necrosis factor-α (TNF-α) play an important role in the pathogenesis of multiple sclerosis (MS). MS is associated with the HLA-DR2, Dw2, DQ6 HLA class II haplotype. Because both LT and TNF-α are encoded in the HLA region, the HLA association of MS may be related to the production of these cytokines. To test this hypothesis, we investigated the production of LT, TNF-α, and interferon-γ (IFN-γ) by CD4⁺ T-cell lines (TCLs) specific for myelin basic protein (MBP) or tetanus toxoid (TT) isolated from MS patients and normal controls. After stimulation with specific antigen but not mitogen, TCLs from HLA-DR2⁺ donors produced significantly more LT and TNF-α, than TCLs from DR2^? donors. In contrast, HLA-DR2⁺ and DR2^? TCLs did not differ in the production of IFN-γ, a cytokine also produced by T cells but not encoded in the HLA region. Increased secretion of LT and TNF-α was unrelated to the specificity (MBP vs TT), MHC restriction (HLA-DR2 vs other DR molecules), or source (MS vs normal) of the TCLs. There was no significant association of the cytokine production with individual LT or TNF-α alleles, indicating that the increased production of these cytokines may be linked to other polymorphic genes in this region. The results suggest that the association of MS with HLA-DR2 implies a genetically determined propensity of T cells to produce increased amounts of LT and TNF-α.     

Targeting ß2 adrenergic receptors regulate human T cell function directly and indirectly

It is well-established that central nervous system activation affects peripheral blood mononuclear cell (PBMCs) function through the release of the catecholamines (Epi) and norepinephrine (NE), which act on ß2-adrenergic receptors (ß2AR). However, most studies have used non-specific stimulation of cells rather than antigen-specific responses. Likewise, few studies have parsed out the direct effects of ß2AR stimulation on T cells versus indirect effects via adrenergic stimulation of antigen presenting cells (APC). Here we report the effect of salmeterol (Sal), a selective ß2AR agonist, on IFN-γ⁺ CD4 and IFN-γ⁺ CD8 T cells following stimulation with Cytomegalovirus lysate (CMVL-strain AD169) or individual peptides spanning the entire region of the HCMV pp65 protein (pp65). Cells were also stimulated with Staphylococcal enterotoxin B. Additionally, we investigated the effect of Epi and Sal on cytotoxic cell killing of transfected target cells at the single cell level using the CD107a assay.The results show that Sal reduced the percentage of IFN-γ⁺ CD4 and IFN-γ⁺ CD8 T cells both when applied directly to isolated T cells, and indirectly via treatment of APC. These inhibitory effects were mediated via a ß2 adrenergic-dependent pathway and were stronger for CD8 as compared to CD4 T cells. Similarly, the results show that Sal suppressed cytotoxicity of both CD8 T and NK cells in vitro following stimulation with Chinese hamster ovary cell line transfected with MICA^*009 (T-CHO) and the human erythromyeloblastoid leukemic (K562) cell line. The inhibitory effect on cytotoxicity following stimulation with T-CHO was stronger in NK cells compared with CD8 T cells.Thus, targeting the ß2AR on lymphocytes and on APC leads to inhibition of inflammatory cytokine production and target cell killing. Moreover, there is a hierarchy of responses, with CD8 T cells and NK cells inhibited more effectively than CD4 T cells.     

Antigen-induced inflammatory mechanical hypernociception in mice is mediated by IL-18

There is pre-clinical evidence that therapies targeting IL-18 might be beneficial in controlling arthropathies, which are accompanied by hypernociception (nociceptor sensitization). In the present study, we addressed the hypernociceptive role of IL-18 in a model of antigen-induced inflammation in mice and its mechanisms. In naïve mice, the intraplantar injection of IL-18 induced dose- and time-dependent mechanical hypernociception, which was inhibited in IFN-γ deficient (−/−) mice, and by the pre-treatment with bosentan (dual endothelin [ET] receptor antagonist), BQ123 (ET_A receptor antagonist) or indomethacin (cyclooxygenase inhibitor). IL-18 hypernociception was unaffected in TNFR1^−/− mice or by the pre-treatment with sIL-15Rα (soluble form of IL-15 receptor), BQ788 (ET_B receptor antagonist) or guanethidine (sympathetic blocker). The ovalbumin (OVA) challenge-induced mechanical hypernociception in immunized mice was inhibited by the pre-treatment with anti-IL-18 antibody or in IL-18^−/− mice. Furthermore, IL-18 induced significant IFN-γ production in the paw skin of naïve mice. The OVA challenge-induced IFN-γ and ET-1 productions were inhibited in IL-18^−/− immunized mice, as well as ET-1 production in IFN-γ^−/− immunized mice. In addition, significant PGE₂ production was detected after IL-18 or ET-1 (via ET_A receptors) injection in naïve mice. Taken together with previous data, these results suggest that IL-18 plays a significant role in antigen-induced inflammatory hypernociception via the production of IFN-γ, ET-1 and PGE₂. Thus, IL-18 and IL-18-downstream mediators demonstrated herein might constitute targets to inhibit antigen-induced inflammatory pain.     

Resistance and susceptibility to experimental autoimmune neuritis in Sprague-Dawley and Lewis rats correlate with different levels of autoreactive T and B cell responses to myelin antigens

Experimental autoimmune neuritis (EAN) is a CD4⁺ T cell-mediated, inflammatory demyelinating disease of the peripheral nervous system (PNS) that serves as a model for Guillain-Barré syndrome (GBS) in humans. Various mouse and rat strains show different susceptibilities to EAN that can be induced by immunization with bovine PNS myelin (BPM) + Freund's complete adjuvant (FCA). We examined PNS-induced T and B cell responses and cytokine protein production as well as mRNA expression to study the mechanisms behind susceptibility to EAN in Lewis rats and resistance in Sprague-Dawley (SD) rats. Lewis rats with EAN have elevated PNS myelin-reactive interferon-γ (IFN-γ) production, TNF-α mRNA expression, and increased B cell responses to PNS myelin antigens, but low PNS myelin-reactive transforming growth factor-β (TGF-β) and interleukin (IL)-10 mRNA expression in lymph node mononuclear cells (MNC). In contrast, resistance to EAN in SD rats is associated with reduced BPM and P2 peptide-reactive IFN-γ production, TNF-α mRNA expression, and suppressed B cell responses to PNS myelin antigens as well as up-regulation of TGF-β and IL-10 mRNA expression. Resistance to EAN is also associated with low-grade inflammation or absence of histological evidence of EAN. These results suggest that differential autoreactive T and B cells responses to PNS myelin antigens are strain specific, and the susceptibility to EAN is related to quantitative rather than qualitative differences in distribution between pro-inflammatory and anti-inflammatory cytokines. J. Neurosci. Res. 54:373–381, 1998. © 1998 Wiley-Liss, Inc.     

Late Anxiety-Like Behavior and Neuroinflammation in Mice Subjected to Sublethal Polymicrobial Sepsis

Sepsis can lead to long-term cognitive changes, including memory and learning deficits, which are known as septic encephalopathy (SE). SE also includes behavioral changes. The underlying mechanism of SE is unknown, and several mechanisms have been proposed. This study investigated late anxiety-like behavior, serum cytokine levels and brain cytokine production in C57BL/6 mice subjected to polymicrobial sepsis induced by sublethal cecum ligature and puncture (CLP). Anxiety-like behavior and locomotor activity were assessed in mice 10 days after sham operation or CLP procedure using the elevated plus maze, contextual fear conditioning, and open field test. Brain and serum concentrations of the cytokines TNF-α, IFN-γ, IL-1β, IL-6, and IL-10 were determined by ELISA. We found that mice subjected to polymicrobial sepsis presented anxiety-like behavior, which was accompanied by increased serum TNF-α and brain TNF-α, IFN-γ, IL-1β, and IL-6 levels, 10 days after the surgical procedure. These findings suggest an involvement of central nervous system inflammatory mediators in the anxiety-like symptoms found in SE.