Sulfasalazine aggravates experimental autoimmune encephalomyelitis and causes an increase in the number of autoreactive T cells.

Sulfasalazine (SASP; 5-(p-(2-pyridylsulfamoyl)phenylazo)salicyclic acid) has beneficial effects on certain inflammatory diseases and has been proposed for clinical trials in multiple sclerosis (MS). We have explored the effects of SASP on actively induced experimental autoimmune encephalomyelitis (EAE) in Lewis rats. SASP was given orally at three different doses from the day of immunization to day 40 post-immunization (p.i.). All doses led to a clinically more protracted disease, increased numbers of T cells infiltrating into the central nervous system (CNS) and to increased numbers of interferon-gamma-secreting cells (IFN-gamma-sc) in the CNS. The effects of SASP treatment on T cell-mediated autoimmunity against CNS myelin and peptides of myelin basic protein (MBP) were measured by IFN-gamma secretion and proliferation by lymph node mononuclear cells in response to these antigens. In SASP-treated rats, increased numbers of IFN-gamma-sc appeared in response to myelin antigens, while the proliferative responses were decreased. We suggest that monitoring cell-mediated immunity with the IFN-gamma-sc method may be relevant for the evaluation of new immunotherapeutic strategies in inflammatory demyelinating diseases. Furthermore, our results demand caution as to clinical trials with SASP in MS.     

T cell immunity and interferon-γ secretion during experimental allergic encephalomyelitis in Lewis rats

An immunospot assay that detects single secretory cells was used to enumerate interferon-γ secreting cells (IFN-γ-sc) in mononuclear cell suspensions from the central nervous system (CNS) and peripheral lymphoid organs after actively induced experimental allergic encephalomyelitis (EAE) in Lewis rats. In the CNS compartment there was a significant increase in the number of IFN-γ-sc preceding the onset of the clinical signs of EAE. Both in rats with EAE and rats immunized with Freund's complete adjuvant (FCA) the number of IFN-γ-sc increased in peripheral lymphoid organs, as compared to non-immunized controls. In view of the potent immunoregulatory effects of IFN-γ, its intra-CNS secretion may play a crucial role for clinicophatological events in EAE.To study the numbers of primed T cells that in response to myelin antigens produced IFN-γ, mononuclear cell suspensions from peripheral lymphoid organs were precultured to allow for antigen uptake, presentation and T cell triggering, followed by enumeration of IFN-γ-sc. T cells responding to a peptide of myelin basic protein (MBP) that previously have been shown encephalitogenic in Lewis rats, appeared initially and were quantitatively dominant over the course of EAE. Later, T cell reactivities to multiple regions of MBP appeared, showing that the concept of immunodominance in EAE is non-absolute and time dependent.Splenocyte cultures from EAE rats exposed to the different antigens showed a reduced number of IFN-γ-sc compared to cultures not exposed to antigen, suggesting an antigwn-induced suppression of T cell effector molecules.     

Cytokines in neuroinflammatory disease: role of myelin autoreactive T cell production of interferon-gamma

Many cytokines must be considered as effector and immunoregulatory molecules in neuroinflammatory diseases such as multiple sclerosis (MS) and experimental allergic encephalomyelitis (EAE). We have studied the potential role of interferon-gamma (IFN-γ) in the pathogenesis of these diseases, since this cytokine has a number of important effects such as macrophage activation, induction of MHC class I and class I antigens, and T cell homing. An immunospot assay that allows enumeration of single cells secreting IFN-γ after short-term culture in vitro of mononuclear cell suspensions has been used. In EAE, increased numbers of IFN-γ-secreting cells (IFN-γ-sc) appear in the central nervous system shortly before onset of clinical signs. Such cells also increased during pharmacologically induced relapse of EAE. In later stages of EAE, memory T cells that produced IFN-γ in response to presented antigen, recognized multiple regions of the myelin basic protein (MBP), showing that (i) myelin autoreactive T cells have the functional ability to produce this cytokine, (ii) the concept of immunodominance as to autoantigen peptide reactivity is non-absolute and time-dependent. In multiple sclerosis (MS) there are increased numbers of IFN-γ-sc among the CSF cells. Also, there are increased numbers of memory T cells, strongly enriched to the cerebrospinal fluid, which upon recognition of several myelin antigens and several MBP peptide stretches, produce IFN-γ. Taken together, the data are consistent with a role for IFN-γ as a key mediator in inflammatory demyelinating diseases.     

Suppression of Autoimmune Neuritis in IFN-γ Receptor-Deficient Mice

Experimental autoimmune neuritis (EAN) is an animal model of the human disease Guillain–Barré syndrome. In this autoimmune inflammatory disease, CD4⁺ T cells mediate demyelination in the peripheral nervous system (PNS). Infiltrating macrophages and T cells as well as cytokines like interferon (IFN)-γ are intimately involved in causing pathogenic effects. To investigate the role of IFN-γ in cell-mediated EAN, IFN-γ receptor-deficient mutant (IFN-γR^−/−) C57BL/6 mice and corresponding wild-type mice were immunized with P0 peptide 180–199, a purified component of peripheral nerve myelin, and Freund's complete adjuvant. IFN-γR^−/− mice exhibited later onset of clinical 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-γR^−/− mice than from wild-type mice on day 24 postimmunization, i.e., at the peak of clinical EAN. At the same time, IFN-γR^−/− mice had less infiltration of inflammatory cells, including macrophages, CD4⁺ T cells, and monocytes, into sciatic nerve tissue and less demyelination. However, numbers of IFN-γ-secreting cells from the spleen were significantly augmented in the IFN-γR^−/− mice, reflecting a failure of negative feedback circuits. The IFN-γR deficiency did not affect the production of anti-P0 peptide 180–199-specific antibodies. These results indicate that IFN-γ contributes to a susceptibility for EAN in C57BL/6 mice by promoting a Th1 cell-mediated immune response and suppressing a Th2 response.     

Interferon-γ Induces Apoptosis and Augments the Expression of Fas and Fas Ligand by Microglia in Vitro

Activation of microglia by interferon-γ (IFN-γ) has been implicated in a number of central nervous system (CNS) inflammatory disease processes. Because IFN-γ has also been shown to play a role in programmed cell death, we investigated its cytotoxicity and its effect on the Fas apoptotic pathway in microglia. Flow cytometry was used to quantify the IFN-γ-mediated apoptotic response and Fas and Fas ligand (FasL) expression in two well-characterized murine microglia cell lines (BV-2 and N9). Nuclear fragmentation, suggestive of apoptosis, was noted within 24 h of incubation of microglia with IFN-γ (10 U/ml). After a 72-h incubation, almost every BV-2 and N9 microglia, but not GL261 glioma cells, underwent cell death and detached from the culture plates. This cytotoxicity occurred even at low IFN-γ concentrations (1 U/ml) and was inhibited by BAF, a pan-caspase inhibitor. Incubation of BV-2 and N9 microglia, but not GL261 glioma cells, with IFN-γ also potentiated the expression of Fas and FasL in a similar dose–response and time-course manner, as seen for the apoptotic response. Whereas Fas expression increased by 100% in both microglia cells, FasL upregulation was more pronounced and increased by as much as 200% in the N9 cells. These findings suggest that in addition to its role as a microglia activator, IFN-γ may also induce apoptosis of microglia, possibly through simultaneous upregulation of Fas and FasL. Interferon-γ modulation of the Fas pathway and apoptosis in microglia may be important in the pathogenesis of inflammatory CNS disease processes.     

Interferon-beta-1a treatment increases CD56 natural killer cells and CD4+CD25+ Foxp3 expression in subjects with multiple sclerosis

Disease modifying effects of interferon (IFN)-β therapy in patients with multiple sclerosis (MS) may be mediated in part through enhanced immunoregulation by the CD56^bright subpopulation of natural killer (NK) cells and by Foxp3+ (not italicized) CD4+CD25+ regulatory T cells (Treg). We found that IFN-β-1a(IM) treatment of relapsing–remitting (RR)MS subjects over 12 months significantly increased both percentage of CD56^bright NK cells and Foxp3 mRNA expression compared to baseline values, untreated RRMS subjects and healthy controls (HC). This striking enhancement of two prominent immunoregulatory pathways lends support to the idea that beneficial effects of IFN-β-1a in MS include control of pernicious autoimmunity.     

Clinical trials of interferons in multiple sclerosis what have we learned?

Although multiple sclerosis (MS) is generally believed to be an immune-mediated disease, conventional therapy with ACTH, corticosteroids, or immunosuppressive drugs is unsatisfactory. Aside from their unpredictable therapeutic effects, these agents are potentially hazardous and can only be given for short periods of time. There is an urgent need for less toxic yet effective immunotherapy, that that can be administered early in the disease and continued indefinitely. Clinical trials of the interferons (IFNs) have not only led to a promising new approach to treatment, but have also stimulated basic research in the immunological mechanisms of underlying disease activity. Administration of IFN-γ promotes exacerbations of MS, whereas recombinant IFN-β has been shown, in controlled clinical trials, to suppress them. Other ongoing studies are likely to provide further information about its long-term therapeutic value. More importantly, laboratory studies performed in conjunction with these clinical trials have provided fresh insights into the pathogenesis of MS by revealing immunoregulatory mechanisms in which endogenous IFN-γ, TNF-α, and other cytokines appear to play central roles. The ‘Decade of the Brain’ may therefore see answers both to the therapeutic dilemma of MS, and to more basic questions about the function of IFNs and other cytokines in activation and regulation of the disease process.     

Effects of γ-interferon on major histocompatibility complex antigen expression and lymphocytic infiltration in the 9L gliosarcoma brain tumor model: implications for strategies of immunotherapy

Effects of γ-interferon (IFN-γ) on immune parameters in the 9L gliosarcoma model were examined. IFN-γ increased class I major histocompability complex (MHC) expression in 9L cells in vitro. In vivo, intratumor injections of IFN-γ led to increased numbers of inflammatory cells within the tumor and class II⁺ mononuclear phagocytes at its periphery, and increased MHC class I or II expression by endothelial and ependymal cells. Class I expression in 9L cells themselves was not increased. This suggests that there may be inhibition of class I induction in vivo for certain cell types, for which immunotherapies based on non-MHC restricted mechanisms may be more effective.     

Treatment with Anti-interferon-γ Monoclonal Antibodies Modifies Experimental Autoimmune Encephalomyelitis in Interferon-γ Receptor Knockout Mice

The role of interferon-γ (IFN-γ) in the pathogenesis of multiple sclerosis and experimental autoimmune encephalomyelitis (EAE) is still controversial. We have studied the function of IFN-γ and its receptor in the EAE model using two different IFN-γ receptor knockout (IFN-γ R^−/−) mouse types: C57Bl/6×129Sv, with a disruption of the IFN-γ receptor cytoplasmic domain, and 129Sv, homozygous for a disrupted IFN-γ receptor gene. Mice were immunized with peptide 40-55 from rat myelin oligodendrocyte glycoprotein. A subgroup of mice was treated with anti-IFN-γ monoclonal antibodies (mAb) on day 8 postimmunization. Clinical scoring and both histological and immunohistochemical studies were undertaken for all groups. We hereby show that treatment with anti-IFN-γ mAb worsened the disease course of 129Sv wild-type mice. However, it decreased the mean daily score in IFN-γ R^−/− 129Sv and the incidence of the disease down to 50% in C57Bl/6×129Sv IFN-γ R^−/− mice. Moreover, after anti-IFN-γ mAb treatment, oxidative stress levels, metallothionein I and II antioxidant protein expression, and apoptoticneuronal death were increased in wild-type mice while decreased in IFN-γ R^−/− mice. These results suggest a putative alternative mechanism of action of this cytokine that works independent of its receptor.     

Alzheimer's β-amyloid peptides induce inflammatory cascade in human vascular cells: the roles of cytokines and CD40

Accumulating evidence suggests that β-amyloid (Aβ)-induced inflammatory reactions may partially drive the pathogenesis of Alzheimer's disease (AD). Recent data also implicate similar inflammatory processes in cerebral amyloid angiopathy (CAA). To evaluate the roles of Aβ in the inflammatory processes in vascular tissues, we have tested the ability of Aβ to trigger inflammatory responses in cultured human vascular cells. We found that stimulation with Aβ dose-dependently increased the expression of CD40, and secretion of interferon-γ (IFN-γ) and interleukin-1β (IL-1β) in endothelial cells. Aβ also induced expression of IFN-γ receptor (IFN-γR) both in endothelial and smooth muscle cells. Characterization of the Aβ-induced inflammatory responses in the vascular cells showed that the ligation of CD40 further increased cytokine production and/or the expression of IFN-γR. Moreover, IL-1β and IFN-γ synergistically increased the Aβ-induced expression of CD40 and IFN-γR. We have recently found that Aβ induces expression of adhesion molecules, and that cytokine production and interaction of CD40–CD40 ligand (CD40L) further increase the Aβ-induced expression of adhesion molecules in these same cells. These results suggest that Aβ can function as an inflammatory stimulator to activate vascular cells and induces an auto-amplified inflammatory molecular cascade, through interactions among adhesion molecules, CD40–CD40L and cytokines. Additionally, Aβ_1–42, the more pathologic form of Aβ, induces much stronger effects in endothelial cells than in smooth muscle cells, while the reverse is true for Aβ_1–40. Collectively, these findings support the hypothesis that the Aβ-induced inflammatory responses in vascular cells may play a significant role in the pathogenesis of CAA and AD.     

Type I interferon receptor signalling is induced during demyelination while its function for myelin damage and repair is redundant

The type I interferons, interferon-beta and alpha (IFN-β, IFN-α), are widely used for the treatment of autoimmune demyelination in the central nervous system (CNS). Their effects on de- and remyelination through the broadly expressed type I IFN receptor (IFNAR), however, are highly speculative. In order to elucidate the role of endogenous type I interferons for myelin damage and recovery we induced toxic demyelination in the absence of IFNAR1. We demonstrate that IFNAR signalling was induced during acute demyelination since the cytokine IFN-β as well as the IFN-dependent genes IRF7, ISG15 and UBP43 were strongly upregulated. Myelin damage, astrocytic and microglia response, however, were not significantly reduced in the absence of IFNAR1. Furthermore, motor skills of IFNAR1-deficient animals during non-immune demyelination were unaltered. Finally, myelin recovery was found to be independent from endogenous IFNAR signalling, indicating a redundant role of this receptor for non-inflammatory myelin damage and repair.     

The effect of interferon β-1b on lymphocyte-endothelial cell adhesion

Interferon β-1b (IFNβ-1b) (Betaseron®) has been recently approved forfor treatment of multiple sclerosis (MS), an inflammatory demyelinating disease of the central nervous system (CNS). The mechanism of action of IFNβ-1b is not understood, but its effect in reducing gadolinium enhanced MRI lesions suggest an effect at the blood brain barrier (BBB). Thus the objective of this study is to examine the effect of IFNβ-1b treatment of endothelial cells (EC) on lymphocyte-EC adhesion, and on the expression of the adhesion molecules (AM) ICAM-1, VCAM and E-selectin induced by IFN-γ, TNF-α, or IL-1β. Primary cultures of human umbilical vein EC (HUVEC) were used which under basal conditions expressed low levels of ICAM-1 but not VCAM or E-selectin. IFNβ-1b (1–1000 IU/ml) had minimal effect on basal expression of AM on HUVEC, but AM could be substantially upregulated by IFN-γ, IL-1β or TNF-α which was associated with a parallel increase in lymphocyte-EC adhesion. The effect of IFNβ-1b on AM expression induced by IFN-γ, IL-1β or TNF-α was slightly additive, and was associated with a modest increase in lymphocyte-EC adhesion. In contrast TGF-β, shown previously to downregulate lymphocyte-EC adhesion, inhibited this adhesion in our experiments. It is concluded that IFN-β does not downregulate the inducible expression of ICAM-1, VCAM or E-selectin on HUVEC and does not inhibit the adhesion of lymphocytes to HUVEC. These findings have implications on the mechanism of action of IFNβ-1b in MS.     

Intracerebral Mycobacterium bovis bacilli Calmette–Guerin infection-induced immune responses in the CNS

To study whether cerebral mycobacterial infection induces granuloma and protective immunity similar to systemic infection, we intracerebrally infected mice with Mycobacterium bovis bacilli Calmette–Guerin. Granuloma and IFN-γ⁺CD4⁺ T cell responses are induced in the central nervous system (CNS) similar to periphery, but the presence of IFN-γIL-17 double-positive CD4⁺ T cells is unique to the CNS. The major CNS source of TNF-α is microglia, with modest production by CD4⁺ T cells and macrophage. Protective immunity is accompanied by accumulation of Foxp3⁺CD4⁺ T cells and PD-L2⁺ dendritic cells, suggesting that both inflammatory and anti-inflammatory responses develop in the CNS following mycobacterial infection.     

Interferon γ- and interleukin-4-secreting cells in multiple sclerosis

Interleukin 4 (IL-4)- and interferon γ (IFNγ)-secreting peripheral blood cells were enumerated by immunospot assay in 13 multiple sclerosis (MS) patients during exacerbations, in 24 patients with progressive multiple sclerosis (CPMS), and in 20 controls. Cells that spontaneously secreted IFNγ were significantly higher in MS patients experiencing an attack (P<0.001) than in controls or in CPMS (P<0.04). IL-4-secreting cell numbers were elevated significantly and to a comparable extent in both MS groups compared to controls. Our finding of increased numbers of IFNγ-secreting cells is in keeping with prior work showing increased IFNγ levels in the circulation prior to and durinng MS attacks and increased release of IFNγ to the supernatant in bulk cultured blood cells from MS patients. What role an increase in IL-4-secreting cells might play in MS is unclear, but it could relate to immune system regulation. Following in vitro exposure to MBP, IFNγ-secreting cell number rose above levels observed in the absence of stimulation in controls and in both MS groups with the rise in acutely exacerbating MS patients being significantly greater than in controls. Our results provide further evidence for reactivity to MBP in MS, the significance of which in terms of pathogenesis remains clouded.     

Determination of interferon-γ in the peripheral blood of multiple sclerosis patients in remission, using a chemiluminescence technique

The plasma from eight patients with multiple sclerosis (MS) whose disease was in remission, was investigated by a chemiluminescence technique for its ability to stimulate the oxidate metabolism of peripheral blood monocytes (PBM). The active fraction identified had a molecular weight between 13 700 and 43 000 Da. Its activity was reduced by incubation at pH 2, pH 4 or pH 6, or by treatment at 56°C for 1–3 h. The activity was also decreased, 58–100%, by prior incubation with antibodies to human interferon-γ (IFN-γ). We suggest that these results indicate that the increased chemiluminescence activity (CL-A) of PBM in MS patients in remission is due mainly to the presence of circulating IFN-γ.     

Rat astrocytes express interferon-γ immunoreactivity in normal optic nerve and after nerve transection

Astrocytes are pivotal components of immune reactions in the CNS. We further support this notion by the localization of the lymphokine interferon-γ (IFN-γ), which plays an important role during immune responses, to astrocytes in rat optic nerve (ON). Astrocytes identified by glial fibrillary acidic protein immunoreactivity were IFN-γ positive in normal and transected ON while oligodendrocytes did not express IFN-γ immunoreactivity. These findings indicate that astrocytes can generate important signals which orchestrate immunoinflammatory responses in the brain.