Effects of pro-inflammatory cytokines on cannabinoid CB1 and CB2 receptors in immune cells.

AIMS: To investigate the regulation of cannabinoid receptors CB1 and CB2 on immune cells by pro-inflammatory cytokines and its potential relevance to the inflammatory neurological disease, multiple sclerosis (MS). CB1 and CB2 signalling may be anti-inflammatory and neuroprotective in neuroinflammatory diseases. Cannabinoids can suppress inflammatory cytokines but the effects of these cytokines on CB1 and CB2 expression and function are unknown. METHODS: Immune cells from peripheral blood were obtained from healthy volunteers and patients with MS. Expression of CB1 and CB2 mRNA in whole blood cells, peripheral blood mononuclear cells (PBMC) and T cells was determined by quantitative real-time polymerase chain reaction (qRT-PCR). Expression of CB1 and CB2 protein was determined by flow cytometry. CB1 and CB2 signalling in PBMC was determined by Western blotting for Erk1/2. RESULTS: Pro-inflammatory cytokines IL-1ß, IL-6 and TNF-α (the latter likely NF-κB dependently) can upregulate CB1 and CB2 on human whole blood and peripheral blood mononuclear cells (PBMC). We also demonstrate upregulation of CB1 and CB2 and increased IL-1ß, IL-6 and TNF-α mRNA in blood of patients with MS compared with controls. CONCLUSION: The levels of CB1 and CB2 can be upregulated by inflammatory cytokines, which can explain their increase in inflammatory conditions including MS.

The endocannabinoid system: a revolving plate in neuro-immune interaction in health and disease.

Studies of the last 40 years have brought to light an important physiological network, the endocannabinoid system. Endogenous and exogenous cannabinoids mediate their effects through activation of specific cannabinoid receptors. This modulatory homoeostatic system operates in the regulation of brain function and also in the periphery. The cannabinoid system has been shown to be involved in regulating the immune system. Studies examining the effect of cannabinoid-based drugs on immunity have shown that many cellular and cytokine mechanisms are modulated by these agents, thus raising the hypothesis that these compounds may be of value in the management of chronic inflammatory diseases. The special properties of endocannabinoids as neurotransmitters, their pleiotropic effects and the impact on immune function show that the endocannabinoid system represents a revolving plate of neural and immune interactions. In this paper, we outline current information on immune effects of cannabinoids in health and disease.

Accelerated thymocyte maturation in IL-12Rß2-deficient mice contributes to increased susceptibility to autoimmune inflammatory demyelination.

IL-12Rß2(-/-) mice, which are unresponsive to IL-12, develop severe experimental autoimmune encephalomyelitis (EAE). The mechanisms for enhanced autoimmunity are incompletely understood. We report that in IL-12Rß2(-/-) mice, thymocytes undergo markedly accelerated maturation. This occurs at the transition from a double positive (DP) to a single positive (SP) phenotype, resulting in higher numbers of CD4 and CD8 SP cells, and to a lesser extent at the transition from double negative (DN) to DP cells. Accelerated maturation is observed in mice injected with anti-CD3 to mimic pre-T-cell receptor stimulation, and also in mice immunized with myelin oligodendrocyte glycoprotein (MOG) peptide to induce EAE.

The protease inhibitor, Bowman-Birk Inhibitor, suppresses experimental autoimmune encephalomyelitis: a potential oral therapy for multiple sclerosis.

Available treatments for multiple sclerosis (MS) require frequent injections and have significant side effects. Proteases generated during inflammation are involved in the induction of tissue damage during inflammatory demyelination in the central nervous system (CNS). The Bowman-Birk Inhibitor (BBI), a soy-derived protease inhibitor with anti-carcinogenic and anti-inflammatory properties, has been shown to be well tolerated in clinical trials for pre-cancerous conditions, such as oral leukoplakia and the inflammatory disease, ulcerative colitis. We hypothesized that BBI may modulate experimental autoimmune encephalomyelitis (EAE), an animal model of MS. The BBI concentrate (BBIC), a soybean extract enriched in BBI, was administered to myelin basic protein (MBP)-immunized Lewis rats by gastric gavage in different treatment regimens, during the induction or the effector phase of disease. BBIC significantly delayed disease onset and suppressed disease severity, clinically and pathologically, in all treatment protocols. Both in vitro and ex vivo, BBIC inhibited MBP-specific proliferation of lymph node cells. BBIC reduced the activity of matrix metalloproteinase (MMP)-2 and -9 in spleen cell supernatants and was detected in the CNS of treated rats. BBIC suppresses EAE, it can be administered orally, and it is safe and relatively inexpensive. It may have a therapeutic role in patients with MS.

Glatiramer acetate induces a Th2-biased response and crossreactivity with myelin basic protein in patients with MS.

Glatiromer acetate (GA) is an approved treatment for multiple sclerosis (MS). The proposed mechanism of action is the induction of GA-specific T cells characterized by protective anti-inflammatory Th2 response. We tested this hypothesis in 11 MS patients treated with GA from 1-19 months. Interferon-gamma and IL-5 (markers of Th1 and Th2 responses respectively) were assayed by ELISA in GA-specific T-cell lines (TCL) supernatants. Th1/Th2 bias was defined based on the ratio of IFN-gamma/IL-5 secretion. Fifty-eight pre-treatment and 75 on-treatment GA-specific TCL were generated. On-treatment mean IL-5 levels in GA-TCL increased significantly, whereas those for IFN-gamma were markedly reduced. Consequently, the ratio of IFN-gamma IL-5 also shifted in favor of a Th2 response. The percentage of GA-TCL classified as Th1 was decreased, whereas those classified as Th2 increased on-treatment as compared to pre-treatment. Some GA-specific TCL, (approximately 25%) generated during treatment secreted predominantly IL-5 in response to MBP and the immunodominant MBP peptide 83-99, indicating that these crossreactive antigens can act as partial agonists for GA-reactive TCL. These results strongly suggest that the mechanism of action of GA in MS involves the induction of crossreactive GA-specific T cells with a predominant Th2 cytokine profile.

Combinatorial peptide libraries and biometric score matrices permit the quantitative analysis of specific and degenerate interactions between clonotypic TCR and MHC peptide ligands.

The interaction of TCRs with MHC peptide ligands can be highly flexible, so that many different peptides are recognized by the same TCR in the context of a single restriction element. We provide a quantitative description of such interactions, which allows the identification of T cell epitopes and molecular mimics. The response of T cell clones to positional scanning synthetic combinatorial libraries is analyzed with a mathematical approach that is based on a model of independent contribution of individual amino acids to peptide Ag recognition. This biometric analysis compares the information derived from these libraries composed of trillions of decapeptides with all the millions of decapeptides contained in a protein database to rank and predict the most stimulatory peptides for a given T cell clone. We demonstrate the predictive power of the novel strategy and show that, together with gene expression profiling by cDNA microarrays, it leads to the identification of novel candidate autoantigens in the inflammatory autoimmune disease, multiple sclerosis.

Molecular mimicry and antigen-specific T cell responses in multiple sclerosis and chronic CNS Lyme disease.

The concept of molecular mimicry provides and elegant framework as to how cross-reactivity between antigens from a foreign agent with self proteins may trigger autoimmune diseases. While it was previously thought that sequence and structural homology between foreign and self proteins or the sharing of T cell receptor (TCR) and MHC-binding motifs are required for molecular mimicry to occur, we have shown that even completely unrelated peptide sequences may lead to cross-recognition by T cells. The use of synthetic combinatorial peptide libraries in the positional scanning format (PS-SCL) together with novel biometric prediction approaches has allowed us to describe the recognition profiles of individual autoreactive T cell clones (TCC) with unprecedented accuracy. Through studies of myelin-specific TCC as well as clones from the nervous system of patients suffering from chronic central nervous (CNS) Lyme disease it has become clear that at least some T cells are more degenerate than previously anticipated. These data will not only help us to redefine what constitutes specific T cell recognition, but also allow us to study in more detail the biological role of molecular mimicry. A recent clinical trial with an altered peptide ligand (APL) of one of the candidate myelin basic protein (MBP) epitopes in MS (amino acids 83-99) has shown that such a modified MBP peptide may not only have therapeutic efficacy, but also bears the potential to exacerbate disease. Thus, we provide firm evidence that the basic principles of cross-recognition and their pathogenetic significance are relevant in MS.

T cells, cytokines, and autoantigens in multiple sclerosis.

In multiple sclerosis (MS), inflammatory demyelination in the central nervous system is thought to be initiated by T cells that recognize myelin antigens. T cells are the main regulators of acquired immunity and are involved in the pathogenesis of several organ-specific autoimmune diseases. This review provides an overview of recent studies on the role of T cells in autoimmune demyelination. Because autoreactive T cells are normally present in the mature repertoire of T cells in the blood and lymphoid organs of MS patients, but also in normal controls, particular attention is devoted to the mechanisms of activation and the functional phenotype of such T cells in patients with MS. The role of cytokines as effector molecules and the main candidate antigens are also discussed.

Mechanisms of immunomodulation by glatiramer acetate.

OBJECTIVE: To define the mechanism of action of glatiramer acetate (GA; formerly known as copolymer-1) as an immunomodulatory treatment for MS. BACKGROUND: The proposed mechanisms of action of GA include 1) functional inhibition of myelin-reactive T cells by human leukocyte antigen (HLA) blocking, 2) T-cell receptor (TCR) antagonism, and 3) induction of T helper 2 (Th2) immunomodulatory cells. In this report, the authors examined the effects of GA on the functional activation of human T-cell clones (TCC) specific for myelin basic protein (MBP) and for foreign antigens. Several questions were addressed: Is the inhibitory effect of GA specific for autoantigens? Is it mediated by blocking the interaction between peptide and HLA molecule? Is GA a partial agonist or TCR antagonist, or does it induce anergy? Does it induce Th2 modulatory T cells? METHODS: The effects of GA on antigen-induced activation of human TCC specific for MBP, influenza virus hemagglutinin, and Borrelia burgdorferi were studied by proliferation and cytokine measurements, TCR downmodulation, and anergy assays. GA-specific TCC were generated in vitro from the peripheral blood of patients and healthy controls by limiting dilution. RESULTS: GA more strongly inhibited the proliferation of MBP, as compared with foreign antigen-specific TCC; in some MBP-specific TCC, the production of Th1-type cytokines was preferentially inhibited. In addition to HLA competition, the induction of anergy, but not direct TCR antagonism, was observed. Numerous GA-specific TCC were generated from the peripheral blood of both MS patients and normal controls, and a fraction of these showed a Th2 phenotype. CONCLUSIONS: This study confirms a preferential inhibitory effect of GA on autoreactive TCC. With respect to cellular mechanisms, although HLA competition appears to play the most important role in functional inhibition in vitro, a direct effect on the TCR may be involved at least in some autoreactive T cells as shown by anergy induction. Although not confirmed at the clonal level, it is demonstrated further that GA induces T cells that crossreact with myelin proteins. GA-specific, Th2-modulatory cells may play an important role in mediating the effect of the drug in vivo.

Association of apolipoprotein E polymorphism to clinical heterogeneity of multiple sclerosis.

In this study we investigated the distribution of apolipoprotein E (APO E) genotypes in sporadic multiple sclerosis (MS) cases and in normal controls. Later onset of chronic progressive MS was observed in patients carrying the epsilon2 allele, whereas APO E alleles were found at similar frequency in MS and in the control population. These findings indicate that clinical heterogeneity, but probably not susceptibility to the disease, is associated to APO E genotypes.

Encephalitogenic potential of the myelin basic protein peptide (amino acids 83-99) in multiple sclerosis: results of a phase II clinical trial with an altered peptide ligand.

Myelin-specific T lymphocytes are considered essential in the pathogenesis of multiple sclerosis. The myelin basic protein peptide (a.a. 83-99) represents one candidate antigen; therefore, it was chosen to design an altered peptide ligand, CGP77116, for specific immunotherapy of multiple sclerosis. A magnetic resonance imaging-controlled phase II clinical trial with this altered peptide ligand documented that it was poorly tolerated at the dose tested, and the trial had therefore to be halted. Improvement or worsening of clinical or magnetic resonance imaging parameters could not be demonstrated in this small group of individuals because of the short treatment duration. Three patients developed exacerbations of multiple sclerosis, and in two this could be linked to altered peptide ligand treatment by immunological studies demonstrating the encephalitogenic potential of the myelin basic protein peptide (a.a. 83-99) in a subgroup of patients. These data raise important considerations for the use of specific immunotherapies in general.

Dipeptidyl peptidase IV (CD26): role in T cell activation and autoimmune disease.

The ectoenzyme dipeptidyl peptidase IV (DP IV; EC 3.4.14.5; CD26) has been shown to play a crucial role in T cell activation. In the present study, we show by flow cytometry and by enzymatic DP IV assay that myelin basic protein (MBP)-specific, CD4+ T cell clones (TCC) derived from patients with multiple sclerosis (MS) express high levels of DP IV/CD26. The enzymatic activity of resting TCC was found to be three to fourfold higher than on resting peripheral blood T cells and close to that of T cells 48 hours after PHA stimulation. The DP IV inhibitors Lys[Z(NO2)]-thiazolidide and Lys[Z(NO2)]-pyrrolidide suppress in a dose-dependent manner DNA synthesis and IFN-gamma, IL-4, and TNF-alpha production of the antigen-stimulated TCC. These data suggest that CD26 plays a role in regulating activation of autoreactive TCC. Further in vivo investigations will clarify, whether the inhibition of the enzymatic activity of DP IV could be a useful tool for therapeutic interventions in MS and/or other autoimmune diseases.

Minimal peptide length requirements for CD4(+) T cell clones--implications for molecular mimicry and T cell survival.

CD4(+) T lymphocytes usually recognize peptides of 12-16 amino acids in the context of HLA class II molecules. We have recently used synthetic peptide combinatorial libraries to dissect in detail antigen recognition by autoreactive CD4(+) T cell clones (TCC). The results of these studies demonstrated that antigen recognition by T cells is highly degenerate and that many cross-reactive ligands can be defined, some of which much more potent than the selecting autoantigen. Based on these observations, we examined the response of a myelin basic protein-specific HLA class II-restricted CD4(+) TCC to truncation variants of optimal ligands. Surprisingly, pentapeptides, tetrapeptides and even tripeptides derived from different segments of the optimal ligands were recognized by the TCC, and some were even more potent than the selecting autoantigen. In addition, these peptides enhanced the survival of the TCC at low concentration. The relevance of this finding was supported by the generation of pentapeptide-specific CD4(+) TCC from peripheral blood lymphocytes. These observations not only change existing views on the length requirements for activation of CD4(+) HLA class II-restricted T cells, but also extend our knowledge about the flexibility of TCR recognition and the potential for cross-reactivity in the immune system.

Contribution of individual amino acids within MHC molecule or antigenic peptide to TCR ligand potency.

The TCR recognition of peptides bound to MHC class II molecules is highly flexible in some T cells. Although progress has been made in understanding the interactions within the trimolecular complex, to what extent the individual components and their amino acid composition contribute to ligand recognition by individual T cells is not completely understood. We investigated how single amino acid residues influence Ag recognition of T cells by combining several experimental approaches. We defined TCR motifs for CD4+ T cells using peptide synthetic combinatorial libraries in the positional scanning format (PS-SCL) and single amino acid-modified peptide analogues. The similarity of the TCR motifs defined by both methods and the identification of stimulatory antigenic peptides by the PS-SCL approach argue for a contribution of each amino acid residue to the overall potency of the antigenic peptide ligand. In some instances, however, motifs are formed by adjacent amino acids, and their combined influence is superimposed on the overall contribution of each amino acid within the peptide epitope. In contrast to the flexibility of the TCR to interact with different peptides, recognition was very sensitive toward modifications of the MHC-restriction element. Exchanges of just one amino acid of the MHC molecule drastically reduced the number of peptides recognized. The results indicate that a specific MHC molecule not only selects certain peptides, but also is crucial for setting an affinity threshold for TCR recognition, which determines the flexibility in peptide recognition for a given TCR.

Lessons from studies of antigen-specific T cell responses in Multiple Sclerosis.

Multiple Sclerosis (MS) is considered a T cell-mediated autoimmune disease of central nervous system myelin. Based on elegant experiments in an animal model of MS, experimental allergic encephalomyelitis (EAE), a number of myelin proteins and peptides derived from these can induce inflammatory demyelinating lesions. Recent studies with transgenic mice expressing human HLA-DR molecules and a myelin basic protein (MBP)-specific T cell receptor as well as data from a phase II clinical trial with an altered peptide ligand based on MBP peptide (83-99) provide convincing evidence that the pathogenetic concepts which largely stem from the above EAE studies are valid in MS, too.

Identification of candidate T-cell epitopes and molecular mimics in chronic Lyme disease.

Elucidating the cellular immune response to infectious agents is a prerequisite for understanding disease pathogenesis and designing effective vaccines. In the identification of microbial T-cell epitopes, the availability of purified or recombinant bacterial proteins has been a chief limiting factor. In chronic infectious diseases such as Lyme disease, immune-mediated damage may add to the effects of direct infection by means of molecular mimicry to tissue autoantigens. Here, we describe a new method to effectively identify both microbial epitopes and candidate autoantigens. The approach combines data acquisition by positional scanning peptide combinatorial libraries and biometric data analysis by generation of scoring matrices. In a patient with chronic neuroborreliosis, we show that this strategy leads to the identification of potentially relevant T-cell targets derived from both Borrelia burgdorferi and the host. We also found that the antigen specificity of a single T-cell clone can be degenerate and yet the clone can preferentially recognize different peptides derived from the same organism, thus demonstrating that flexibility in T-cell recognition does not preclude specificity. This approach has potential applications in the identification of ligands in infectious diseases, tumors and autoimmune diseases.

Molecular mimicry and multiple sclerosis--a possible role for degenerate T cell recognition in the induction of autoimmune responses.

Multiple sclerosis is an inflammatory demyelinating disease of the central nervous system. The etiology is unknown, but several lines of evidence support the hypothesis that the pathogenesis is mediated by autoreactive T lymphocytes. Molecular mimicry has been proposed as a possible mechanism for the development of an autoimmune response to myelin antigens. According to this model, an immune reaction to self antigens could be initiated by T cells that cross-react with infectious agents that "mimic" the autoantigen, i.e. they share immunologic epitopes. It was previously thought that, in order for a cross-reaction of T cells to two different antigens to occur, a substantial amino acid sequence homology between the two antigens was required. More recent studies on the basic mechanisms of T cell antigen recognition have shown that, at least for some T cell clones, antigen recognition is more "degenerate" and sequence homology is not required for crossreactivity to occur. This article reviews the relevance of these recent advances in basic T cell receptor immunology to the occurrence of autoimmunity in the central nervous system.

Exploring immunological specificity using synthetic peptide combinatorial libraries.

The definition of epitopes for human B and T cells is fundamental for the understanding of the immune response mechanism and its role in the prevention and cause of human disease. This understanding can be applied to the design of diagnostics and synthetic vaccines. In recent years, the understanding of the specificity of B and T cells has been advanced significantly by the development and use of combinatorial libraries made up of thousands to millions of synthetic peptides. The use of this approach has had four major effects: first, the definition of high affinity ligands both for T cells and antibodies; second, the application of alternative means for identifying immunologically relevant peptides for use as potential preventive and therapeutic vaccines; third, a new appreciation of the requirements for TCR interactions with peptide-MHC complexes in immunogenicity; fourth, the establishment of new principles regarding the level of cross-reactivity in immunological recognition.

Molecular mimicry and multiple sclerosis: degenerate T-cell recognition and the induction of autoimmunity.

Various mechanisms have been proposed for the initiation of autoimmune responses by autoreactive T-cell clones. One of these, the molecular mimicry hypothesis, postulates that myelin-reactive T-cell clones are activated by foreign antigens. Until recently, sequence homology between self- and foreign antigens was considered necessary for cross-recognition to occur in multiple sclerosis. This article reviews current progress in T-cell receptor immunology that led to modify this view and proposes a role for degenerate T-cell antigen recognition in the induction of autoimmunity.