Deming Sun

Expression of chemokine genes in rat glial cells: The effect of myelin basic protein-reactive encephalitogenic T cells

Chemokine gene expression and chemokine activity appear to be major components of the immunopathological processes of inflammation and autoimmunity. To initiate an investigation of the role of chemokines in the pathogenesis of autoimmune inflammatory demyelination, we examined the expression of mRNA transcripts encoding four prominent chemokines, IP‐10, MIP‐1α, MCP‐1, and RANTES, in encephalitogenic rat MBP‐reactive T cells, astrocytes, and microglia. Astrocytes and microglia, whether as lines or as freshly isolated cells, did not constitutively express IP‐10 and MCP‐1 mRNA but could be induced with LPS to also produce MIP‐1α and RANTES. MBP‐reactive T cells were induced with MBP to produce abundant levels of MIP‐1α, MCP‐1, and RANTES mRNA in different temporal profiles but did not express IP‐10 mRNA. In an MHC‐II restricted fashion, the antigen‐activated MBP‐reactive T cells also induced glial cells to express all four chemokines, with the chemokine gene expression greatest following T‐cell interactions with MHC‐compatible glia. Treatment of glial cells with TNF‐α and IFN‐γ induced only IP‐10, indicating that the expression of chemokine genes other than IP‐10 requires a combination of different cytokines or direct cell‐cell contact between T cells and glia. Quantitative assays revealed that activated astrocytes, the dominant glia of the CNS, express higher levels of chemokine transcripts than transcripts of the major proinflammatory cytokines TNF‐α and IFN‐γ. These results underscore the prominent but complex expression of chemokines by cellular component of inflammatory demyelinating lesions. J. Neurosci. Res. 48:192–200, 1997.

CITRULLINATED MYELIN BASIC PROTEIN INDUCES EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS IN LEWIS RATS THROUGH A DIVERSE T CELL REPERTOIRE

An increased proportion of citrullinated MBP (MBP-C8) occurs in the brains of multiple sclerosis (MS) patients. In this study, MBP-C8 from guinea pig (GP) brains was isolated and found encephalitogenic in Lewis rats upon immunization. An encephalitogenic T cell line selected with MBP-C8 preferentially reacted with MBP-C8 over unmodified MBP. This T cell line responded weakly to the dominant encephalitogenic epitope, GP-MBP peptide 70-88, and did not display restricted TCR beta-chain usage (such as Vbeta88.2). The distinctive features of MBP-C8 were also demonstrated by its ability to reinduce active EAE in 70% of rats which had recovered from unmodified MBP induced EAE. These findings raise the possibility that citrullinated MBP may elicit a different pathogenic T cell repertoire for the recurrent phases of inflammatory demyelination.

Inhibition of experimental allergic encephalomyelitis in the Lewis rat by paclitaxel.

Experimental allergic encephalomyelitis (EAE), an animal model for multiple sclerosis (MS), is useful for preclinical testing for agents to be considered for treatment for this human demyelinating disease. Microtubules in lymphocytes play an important role in the cascade of human T cell activation, and paclitaxel (PTX), a microtubule stabilizer, can inhibit T cell function. A new formulation of micellar PTX, free of Cremophor and ethanol, was tested for its effect on the induction of EAE in Lewis rats. Adoptive EAE was induced with an encephalitogenic T cell line activated with guinea pig myelin basic protein (GP MBP) peptide 68-88. PTX (10 mg/kg) was administered 24 and 72 h after cell transfer. The clinical signs, fulminating in controls, were completely blocked by PTX, but mild CNS inflammation remained unaltered. A similar dose of PTX, given on days 6 and 8 to animals developing active EAE after immunization with GP MBP peptide 68-88 in complete Freunds adjuvant, greatly reduced the severity of paralysis and delayed the onset of disease by 8-9 days. Marked weight loss and severe toxicity were noted with higher and more prolonged administration. In vitro micellar PTX inhibited activation of encephalitogenic T cells by both specific antigen and mitogen. Lower doses and longer treatment programs may provide effective treatment with acceptable adverse effects with this agent in the treatment of inflammatory demyelinating disease.

Reciprocal stimulation between TNF-α and nitric oxide may exacerbate CNS inflammation in experimental autoimmune encephalomyelitis

Nitric oxide (NO) and TNF-alpha are both highly active pleotypic modulators of cell function that are abundantly generated during inflammation. Experiments in animal systems have linked the generation of NO and TNF-alpha to autoimmune pathogenesis, and blockade of either NO or TNF-alpha has been shown to impede disease development. In this study, we show that NO and TNF-alpha can act mutually to stimulate each others production. While IFN-gamma alone induces NO release from microglia, astrocytes are provoked into significant NO production only by a combination of IFN-gamma and TNF-alpha. Since both TNF-alpha and NO are abundantly generated during T-glial cell interaction, we asked whether and how NO affects TNF-alpha production. Using an in vitro system in which TNF-alpha secretion is induced in MBP-reactive T cells by co-culture with syngeneic astrocytes, we observed that the efficiency of TNF-alpha secretion was markedly increased, in a dose-dependent fashion, by addition of micromolar concentrations of a chemical generator of NO donor, sodium nitroprusside (SNP). Similarly, low concentrations of SNP significantly enhanced the IL-2 dependent growth of MBP-reactive T cells. These results suggest that autoimmune pathogenesis initiated by inflammatory responses within the CNS may result in part from a vicious cycle in which TNF-alpha and NO mutually provoke each others production.

Nonactivated astrocytes downregulate T cell receptor expression and reduce antigen-specific proliferation and cytokine production of myelin basic protein (MBP)-reactive T cells

Astrocytes express variable levels of MHC class II antigens depending on their activation status or exposure to certain cytokines, notably IFN-gamma. When they are induced to express higher surface densities of MHC class II molecules, astrocytes are capable of stimulating syngeneic myelin basic protein (MBP)-reactive T cells to proliferate at a modest rate and to secrete proinflammatory cytokines, such as TNF-alpha, in response to antigen. In the present investigation evidence is presented that uninduced astrocytes, whether fresh or established as clones, on which surface MHC class II molecules are expressed at a very low density, promote an antigen-dependent reduction of TCR on the surface of syngeneic T cells. Accompanying this effect on the TCR is an induction of T cell hyporeactivity and little or no production of proinflammatory cytokines. These observations suggest that the ability of the astrocyte, through varying their surface MHC class II molecules, can control the effect of antigen-induced T cell responses. In their normal state of low MHC II expression astrocytes are expected to induce no or partial, rather than full, activation of autoreactive T cells that enter the CNS, resulting in T cell hyporeactivity. Since astrocytes usually diminish the production of proinflammatory cytokines by T cells that enter the CNS, the status and control of MHC class II expression on astrocytes should be important determinants of the suppression or enhancement of in situ immune responses in the CNS.

The role of regulatory T cells in Lewis rats resistant to EAE

Adult Lewis (LEW) rats are highly susceptible to experimental autoimmune encephalomyelitis (EAE), induced actively by immunization with guinea pig (GP) myelin basic protein (MBP) in complete Freunds adjuvant or adoptively transferred with activated T lymphocytes reactive to GP MBP peptide 68-88. Once LEW rats recover from active EAE or when given MBP in incomplete Freunds adjuvant (IFA), they become resistant to further attempts to induce active or passive EAE. In this study, we examined whether such EAE-resistant rats after MBP-IFA immunization have reduced frequencies of MBP-reactive T cells, whether these T cells are anergized, and whether the activity of regulatory T cells is increased to the event that they prevent activation of MBP-specific T cell subpopulations. By limiting dilution analyses (LDA) of unfractionated splenic T cells, the levels of MBP-reactive T cells in EAE-resistant rats appeared to be approximately 5% of the levels in EAE-susceptible rats. However, a subsequent analysis of CD4+ enriched T cell populations, depleted of the CD8 subset, showed similar frequencies of MBP-reactive cells in susceptible and resistant LEW rats. Not only were the frequencies on LDA altered by suppressor cells, but also LDA comparisons based on cell proliferation and cytokine production as indicators of MBP reactive cell frequencies gave markedly different results. We conclude that MBP-reactive T cells in this model of EAE-resistant LEW rats are hyporeactive to MBP as the result of an increased activity of a regulatory subset of CD8+ T cells. These results also demonstrate that the quantitation of MBP-reactive CD4+ T cells by LDA is strongly influenced by the presence of functionally antagonistic CD8+ T cells, which cause an underestimation of responder T cell frequencies, and by the method of detecting T cell reactivity.

Regulatory T cells in experimental allergic encephalomyelitis. II. T cells functionally antagonistic to encephalitogenic MBP-specific T cells show persistent expression of fasL.

Naive LEW rats and animals that have recovered from active or adoptive EAE contain a subset of T cells that inhibit EAE in secondary recipients and are lytic for MBP‐reactive T cell lines in culture. Here we explore various features of these regulatory T cell populations (RTC) with respect to (1) their frequency in animals following immunization with syngeneic MBP‐reactive cell lines, (2) their ability to inhibit proliferative responses by MBP‐reactive cell lines in culture, (3) their ability to lyse MBP‐specific target cells, and especially (4) their prolonged expression of high levels of FasL following activation in culture correlating with their lytic effects on A20, a FasL‐sensitive mouse lymphoma cell line. Inhibition studies indicate that mAbs specific for MHC class I and MHC class II molecules inhibit lysis of syngeneic MBP‐reactive target T cells, soluble Fas protein shows some inhibition, but none of these agents inhibits the lytic effects of activated RTC on the A20 cell line. J. Neurosci. Res. 58:357–366, 1999.

Alanine-substituted peptide ligands differ greatly in their ability to activate autoreactive T-cell subsets specific for the wild-type peptide

Alanine-substituted peptide ligands (APLs) have the potential to reduce or block autoreactive T-cell activation. Most previous investigations aimed at either identification of the amino acid residue within a peptide ligand that is critical for T-cell activation or characterization of inhibitory APLs have analyzed the effects of APLs on one, or a limited number, of T-cell lines. In this study, we compared the effects of a panel of peptides on the proliferative and activation responses of one T-cell line as well as the effects of one peptide on the responses of a panel of T-cell lines. This study reveals that the T cells that comprise the T-cell population that responds to a specific peptide are heterogeneous in that an APL may fail to induce a response in some of these T cells although it is capable of inducing a response in the others. Moreover, APLs can induce T-cell activation, in terms of production of IL-2 and/or TNF-alpha, in the absence of appreciable cell proliferation. Indeed, despite being poor stimulators in proliferation assays, most APLs readily induce production of TNF-alpha. Our results demonstrate that the net outcome of APL treatment in vivo represents the sum of diverse effects, which may not be revealed completely by limited and randomly chosen in vitro assays.

Quantitative assessment of myelin basic protein-reactive T cell entry to the central nervous system by using oligonucleotide probes complementary to VDJ junctional sequences of rat TCR β-chain

The VDJ junctional region represents the most diverse part of the antigenic TCR. We have previously reported that of 200 sequenced TCR beta-chains of rat MBP-reactive T cells, rarely did two share sequence homology over the entire CDR3 region. In this study, we demonstrate that sequences of the TCR CDR3 region are excellent clonotypic markers for rat MBP-reactive T cell clones and oligonucleotide probes complementary to the CDR3 region of three T cell clones specifically recognized the TCR from which they were derived, but failed to recognize syngeneic T cells that express a similar TCR beta-chain or T cells that share both V beta and J beta usage. To explore this observation, we determined the ability of MBP-reactive T cell clones to enter the CNS. We were able to show that some MBP-reactive T cell clones have an augmented ability to enter the CNS and that fully-activated T cells have a higher penetrating activity than their less-activated T cell counterparts.

Nitric oxide and TBF-alpha exacerbate each other's production in CNS inflammation

To investigate the regulatory effect of nitric oxide via constitutive nitric oxide synthase (cNOS) in autoimmtme spinal cord injury, we examined the expression of eNOS in the spinal cord of rats with EAE and compared iNnS immunoreactivity. Also we analysed the relationship between eNOS+ cells and apoptotic cells in EAE lesions. cNOS immunoreacfivity in the spinal cords was detected in some neurons, a few astrocytes and some vesse ls at the peak stage of EAE, but its expression was not detected in the normal rat spinal cords. However, iNnS+ cells were mainly localized in perivascular cuffing as well as some astrocytes at the peak stage. By double labeling method, apeptotie cells were dispersed on the spinal cord with EAE, and these cells were in contact with eNOS+cells including neurons and astrocytes. These findings sugges t that NO via eNOS from astrocytes and neurons play an important role in eliminating inflammatory cells via apoptosis leading to central nervous sys tem repair. 284 Primary Role for Macrophages/Microglia in Mediating a Delayed Onset Neuroinflammatory Disease in GFAP-TNF-a Transgenic Mice A,K. Stalder. M. Carson, V.C. Ascnsio, TheScrippsResearchlnstinae, USA, E. Masliah, H. Powell, University of California, San Diego, USA, I.L. Campbell, The Scripps Research Institute, USA