Interferon-γ secretion by in vivo activated cytotoxic T lymphocytes from the blood and cerebrospinal fluid during mumps meningitis

Functional studies of cerebrospinal fluid T lymphocytes during acute viral infections of the nervous system are rare. Recently, we had the opportunity to investigate the requirments for interferon-γ (IFN-γ) production of human in vivo activated (primary) cytotoxic T lymphocytes (CTL) generated during acute viral meningitis. Two HLA-B7-restricted, CD4⁻, CD8⁺ CTL clones from cerebrospinal fluid of one patient with mumps meningitis were studied. Although lytic activity was restricted by HLA-B7, the clones produced similar amounts of IFN-γ when stimulated with HLA-matched and mismatched mumps virus-infected target cells. In addition, peripheral blood mononuclear cells of infected patients secreted significant amounts of IFN-γ when incubated with autologous or allogeneic (HLA-A/B-mismatched) mumps virus-infected target cells. T cells capable of lytic activity and IFN-γ secretion could only be isolated from venous blood during the initial phase of the infection. We suggest that the ability of human in vivo activated CTL to secrete INF-γ early during the course of inflammation and in a HLA-unrestricted fashion is important for the elimination of viruses invading the central nervous system.     

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.     

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.     

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.     

L-Selectin Expression Affects T-Cell Circulation Following Isoproterenol Infusion in Humans

β-Adrenergic receptor agonists have been shown to affect leukocyte migration. This study examined the expression of cellular adhesion molecules on lymphocyte, monocyte, and granulocyte distribution following an infusion of isoproterenol (20 and 40 ng/kg/min for 15 min each) in 12 healthy subjects. Leukocyte populations and adhesion molecule expression were determined via flow cytometry. Isoproterenol led to an expected lymphocytosis and leukocytosis. L-selectin expression varied across leukocytes and influenced cell trafficking in response to isoproterenol. Approximately 60% of CD8⁺T-cells expressed L-selectin (CD8⁺CD62L⁺) and these cells showed no appreciable response to isoproterenol. In contrast, CD8⁺CD62L⁻cells showed a robust increase in number and distribution of approximately 100% over baseline (p’s < .001). Across CD4⁺T-helpers, L-selectin was expressed on approximately 86% of cells. CD4⁺CD62L⁺cells decreased in number and distribution (p’s < .001) with isoproterenol, while CD4⁺CD62L⁻cells showed a modest increase (p’s < .05). In contrast to lymphocytes, nearly all monocytes and granulocytes expressed L-selectin; these cells increased and decreased respectively in response to isoproterenol (p’s < .05). CD11a (the β₂-integrin LFA-1) was expressed on >95% of all leukocytes and these data were thus similar to the overall leukocytosis data. CD54 (ICAM-1) was expressed on approximately 60% of mixed lymphocytes and was unchanged in response to isoproterenol. The findings indicate that L-selectin expression influences T-cell trafficking in response to β-adrenergic stimulation and help further illuminate catecholamine-mediated sympathetic and immune interactions.     

Restraint stress modulates virus specific adaptive immunity during acute Theiler’s virus infection

Multiple sclerosis (MS) is a devastating CNS disease of unknown origin. Multiple factors including genetic background, infection, and psychological stress affect the onset or progression of MS. Theiler’s murine encephalomyelitis virus (TMEV) infection is an animal model of MS in which aberrant immunity leads to viral persistence and subsequently results in demyelination that resembles MS. Here, we examined how stress during acute TMEV infection altered virus-specific cell mediated responses. Using immunodominant viral peptides specific for either CD4⁺ or CD8⁺ T cells, we found that stress reduced IFN-γ producing virus-specific CD4⁺ and CD8⁺ T cells in the spleen and CD8⁺ T cells CNS. Cytokine production by cells isolated from the CNS or spleens following stimulation with virus or viral peptides, indicated that stress decreased both type 1 and type 2 responses. Glucocorticoids were implicated in the decreased T cell function as the effects of stress were partially reversed by concurrent RU486 administration but mimicked by dexamethasone. As T cells mediate viral clearance in this model, our data support the hypothesis that stress-induced immunosuppression may provide a mechanism for enhanced viral persistence within the CNS.     

T Lymphocyte Subsets and Cytokines in Rats Transplanted with Adipose-Derived Mesenchymal Stem Cells and Acellular Nerve for Repairing the Nerve Defects

Objective

The aim of this study was to explore the immunity in rats transplanted with adipose-derived mesenchymal stem cells (ADSCs) and acellular nerve (ACN) for repairing sciatic nerve defects.

Methods

ADSCs were isolated from the adipose tissues of Wistar rats. Sprague-Dawley rats were used to establish a sciatic nerve defect model and then divided into four groups, according to the following methods : Group A, allogenic nerve graft; Group B, allograft with ACN; Group C, allograft ADSCs+ACN, and Group D, nerve autograft.

Results

At the day before transplantation and 3, 7, 14, and 28 days after transplantation, orbital venous blood of the Sprague-Dawley rats in each group was collected to detect the proportion of CD3⁺, CD4⁺, and CD8⁺ subsets using flow cytometry and to determine the serum concentration of interleukin-2 (IL-2), tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) using enzyme-linked immunosorbent assay (ELISA). At each postoperative time point, the proportion of CD3⁺, CD4⁺, and CD8⁺ subsets and the serum concentration of IL-2, TNF-α, and IFN-γ in group C were all near to those in group B and group D, in which no statistically significant difference was observed. As compared with group A, the proportion of CD3⁺, CD4⁺, and CD8⁺ subsets and the serum concentration of IL-2, TNF-α, and IFN-γ were significantly reduced in group C (p<0.05).

Conclusion

The artificial nerve established with ADSCs and ACN has no obvious allograft rejection for repairing rat nerve defects.     

Characterization of a unique T-cell clone established from a patient with HAM/TSP which recognized HTLV-I-infected T-cell antigens as well as spinal cord tissue antigens

Five T-cell clones reactive to autologous HTLV-I-infected T-cells (KODA-TV) were established from peripheral blood lymphocytes of a HAM/TSP patient (KODA) by the limiting dilution method. All the clones showed CD3⁺, CD4⁺ and CD25⁺ surface markers and expressed αβ⁺ T-cell receptors to recognize KODA-TV antigens. One of the five T-cell clones (KODA-408) was infected with HTLV-I but the remaining four clones (KODA-400, 404, 405 and 409) were free of HTLV-I infection. KODA-408 recognized both KODA-TV and spinal cord antigens, the latter being extracted from autopsy tissues of a HTLV-I seronegative donor. KODA-408 did not recognize either alloantigens of peripheral blood mononuclear cells extracted from unrelated HTLV-I seronegative donors or purified human myelin basic protein. KODA-408 T-cell clone produced a considerable amount of TNF-aβ, IFN-γ, and IL-6. The CDR3 motif of KODA-408 T-cell receptor showed a unique sequence CASSAGQS of Vβ8-Dβ-Jβ1.5. These results indicated that HAM/TSP CD4⁺ T-cells were polyclonally activated by HTLV-I infection and antigenic stimulation. The T-cell repertoire shaped by HTLV-I infection included T-cells which recognized HTLV-I-infected T-cell antigens as well as spinal cord antigen in particular.     

Lymphokine mRNA expression in the spinal cords of Lewis rats with experimental autoimmune encephalomyelitis is associated with a host recruited CD45R hi/CD4 population during recovery

To evaluate CD4⁺ T cell subpopulations involved in the induction and recovery from experimental autoimmune encephalomyelitis (EAE), the CD45R phenotype and lymphokine mRNA profile was evaluated for encephalitogenic CD4⁺ T cell lines in vitro and compared to CD4^* T cells islated from the spinal cord of Lewis rats with EAE were > 90% of the myelin basic protein (MBP)-specific T cell lines and clones that adoptively transferred EAE were > 90% CD4⁺ and > 90% CD45R lo. A time course of EAE disease progression was monitored as a function of the percentage of CD45R hi/CD4⁺ T cells isolated from the spinal cords of diseased animals. The majority of CD4⁺ T cells found in the central nervous system during the early phase of passive EAE were CD45R lo (the same as the encephalitogenic lines/clones). A large increase of the CD45R hi/CD4⁺ T cells (up to 45%) was observed during the peak and recovery phases of EAE. Lymphokine mRNA production was analyzed from antigen-stimulated MBP-specific lines, and from spinal cord lymphocytes isolated from rats with EAE. The BP-specific lines produced Th1 lymphokines (IL-2, IFN-γ, and TNF-α), while the spinal cord lymphocytes produced the same Th1 lymphokines as well as IL-4 and IL-10. The CD45R hi/CD4⁺ T cells isolated from the spinal cords were larger and expressed more lymphokine RNA per cell than the CD45R lo/CD4⁺ T cells. The encephalitogenic cells (CD45R hi/CD4⁺ T detected in the spinal cords of rats with a fluorescent dye and by allelic transfers and all of the CD45R hi/CD4⁺ lymphocytes found in the spinal cells were found to be host recruited. Thus it appears that the CD45R hi/CD4⁺ lymphocytes found in the spinal cord represent a host-recruited, activated cellular infiltrate that increased in number in the recovery phase of EAE and synthesized both Th1 and Th2 lymphokines.     

Human CD8 TCR-αβ and TCR-γδ cells modulate autologous autoreactive neuroantigen-specific CD4 T-cells by different mechanisms

To investigate the regulatory interactions among autologous T-cells during the course of multiple sclerosis (MS), proteolipid protein peptide-specific CD4⁺ T-cell clones (TCCs) were irradiated and used as immunogens to stimulate purified populations of autologous CD8⁺ TCR-αβ⁺ and TCR-γδ⁺ T-cells isolated from the peripheral blood of MS patients, patients with other non-inflammatory neurological diseases, and healthy blood donors. The resulting blasts were expanded in the presence of hIL-2 and then cloned by limiting dilution. Two different groups of CD8⁺ TCCs were revealed. A first group of CD8⁺ TCCs recognized autologous CD4⁺ T-cells based in their TCRVβ structures (anti-idiotypic responsiveness). A second group of CD8⁺ TCCs recognized Ag activated autologous CD4⁺ TCCs irrespective of their Ag specificity or TCRVβ expression (anti-ergotypic responsiveness). Both groups showed MHC class I restricted cytotoxicity against CD4⁺ T-cells and were able to secrete IFN-γ, TNFα/β and TGF-β. TCR-γδ⁺ TCCs isolated in response to stimulation with autologous peptide-specific CD4⁺ TCCs showed only anti-ergotypic cytotoxicity, which was not inhibited by anti-MHC class Ia monoclonal antibodies. Moreover, they were able to secrete IFN-γ and TNFα/β, but not TGF-β. These data demonstrate that regulatory mechanisms among human autologous T-cells can be mediated by cytolytic interactions or by the release of specific cytokines. Furthermore, they provide evidence that CD8⁺ TCR-αβ⁺ and TCR-γδ⁺ cells differ in their patterns of recognition and in their abilities to modulate the immune response mediated by autologous autoreactive CD4⁺ T-cells.     

Endogenous gamma interferon produced in central nervous system by systemic infection infection with Theiler's virus in mice

Theiler's virus GD VII strain causes acute encephalomyelitis by intracerebral inoculation. We established acute encephalomyelitis in mice by the intravenous (i.v.) inoculation of Theiler's virus GD VII strain. Replication of Theiler's virus injected i.v. could be observed in both the brain and spinal cord of mice, and interferon (IFN)-γ could be detected in the extracts of brain and spinal cord in parallel with viral replication. Furthermore, by the injection of anti-IFN-γ monoclonal antibody (mAb) on Day 1 post-infection (p.i.), mortality and virus titres in the spinal cord increased compared with the control mice treated with normal rat globulin. The histological exacerbation of inflammation was observed in spinal cord of anti-IFN-γ mAb-treated mice. These results indicate that endogenous IFN-γ, produced locally in the brain and spinal cord of mice through both antiviral action and anti-inflammatory action of IFN-γ in central nervous system, plays an important role in Theiler's virus infection.     

Lymphocyte traffic changes induced by monolateral vagal denervation in mouse thymus and peripheral lymphoid organs

In this report we show that after monolateral vagal denervation (vagotomy), performed at the cervical level, a transient effect, lasting about 24 h, was produced on lymphocyte release from mouse thymus to peripheral lymphoid organs (spleen and lymph nodes). Labelling thymocytes in situ with fluorescein isothiocyanate (FITC) we note that the export of immature cells, CD4⁺CD8⁺, double positive (DP), and double negative, CD4⁻CD8⁻ (DN), from the thymus was consistently increased 24 and 48 h after vagotomy. Double staining with anti-L3T4 (CD4) and anti-mouse CD8α showed that the number of DP and UN cells was significantly higher in both spleen and lymph nodes of vagotomized mice compared to controls (sham-operated), whereas the percentage of CD4⁺CD8⁻ and CD8⁺CD4⁻, single positives (SP), was decreased. Considering thymic cellularity and apoptotic values, we exclude the non-specific effect of stress and suggest that this phenomenon could be in part due to a transient lack of the facilitating influence exerted by vagal efferent fibers on lymphocyte traffic at the cortico-medullary junction of the thymic gland, where mature cells, SP, leave the thymus to enter systemic circulation.     

Local immune responses in the rat cerebral cortex after middle cerebral artery occlusion

This study describes local immune responses in cerebral ischemia induced by permanent occlusion of the middle cerebral artery (MCAO) in the rat. The temporal and spatial pattern of leukocyte infiltration was characterized immunocytochemically using monoclonal antibodies against CD5, a pan T cell marker, against CD4 and CD8 for subtyping of T lymphocytes, and ED1, a marker for macrophages. CD5⁺ T cells were present in some animals on the pial surface at day 1 and with increasing numbers mainly at the edges of the infarcts all days 3 and 7. By day 14 their number had significantly decreased. Subtyping of T lymphocytes revealed that CD4⁺ helper/inducer T cells were rare, while CD8⁺ lymphocytes were abundant. Moreover, CD8⁺ lymphocytes outnumbered CD5⁺ T cells indicating the presence of CD5⁻/CD8⁺ natural killer (NK) cells. ED1⁺ macrophages primarily infiltrated the core of the infarct starting on day 1. Infiltrating leukocytes expressed leukocyte function associated antigen-1 and MHC class I and II antigens. Early after infarction, increased expression of the intercellular adhesion molecule-1 was found on vessel and leukocytes. In conclusion, this study shows that lymphocytes enter the nervous system not only in autoimmune diseases, but also in response to primarily ‘non-immune’ neuronal damage such as stroke.     

Transforming growth factor-β1 inhibits tumor necrosis factor-α/lymphotoxin production and adoptive transfer of disease by effector cells of autoimmune encephalomyelitis

We previously reported that the CD4⁺ suppressor cells (Ts) that regulate recovery of Lewis rats from experimental autoimmune encephalomyelitis (EAE) produce transforming growth factor-β (TGF-β). We also reported that TGF-β downregulates interferon-γ (IFN-γ), but not interleukin-2 (IL-2) production, by the CD4⁺ effector T cells (Te) that mediate EAE. We now report that TGF-β also inhibits the production of tumor necrosis factor/lymphotoxin (TNF/LT) by EAE effector cells. When activated in vitro with myelin basic protein (MBP), Te produced TNF/LT, as measured using a WEHI 164 cytotoxicity assay. The specificity of cytokine action was demonstrated using neutralizing antibodies to TNF/LT. When added to the Te + MBP cultures, TGF-β inhibited TNF/LT production in a dose-dependent fashion. Moreover, neutralizing anti-TGF-β antibodies augmented TNF/LT production in the Te + MBP cultures. We also confirm that TGF-β inhibits adoptive transfer of EAE. In contrast, murine IL-10 only partially inhibited TNF/LT and IFN-γ production by Te. We conclude that TGF-β production by Ts plays a major role in recovery from EAE in the Lewis rat by inhibiting TNF/LT and IFN-γ production by the effector cells that mediate EAE.     

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.