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Altered peptide ligand modulation of experimental allergic encephalomyelitis: Immune responses within the CNS
Abstract
An altered peptide ligand (analog) of the encephalitogenic epitope of proteolipid protein residues 139-151 (p139-151) in which residues 144 and 147 are substituted with leucine and arginine, respectively (LR), protects from clinical but not histological experimental allergic encephalomyelitis (EAE). To understand in situ events associated with this protection, T cells from brains of mice immunized with either native p139-151, the analog LR or a combination of the two were isolated and characterized. High proportions of cells from co-immunized mice (38%) and LR-immunized mice (58%) reacted to both p139-151 and LR, whereas fewer cells from p139-151 immunized mice (7%) were cross-reactive. T cell clones derived from brains of LR- and co-immunized mice were also cross-reactive in vitro. By reverse transcriptase-based polymerase chain reaction, higher levels of TGF-beta mRNA, and lower levels of TNF-alpha and IFN-gamma mRNA were found in the central nervous system (CNS) tissue of LR and co-immunized mice. Immunohistochemistry demonstrated greater TGF-beta immunoreactivity in CNS inflammatory foci in co-immunized and LR-immunized mice. There were no significant differences in CD4+ or CD8+ cell infiltrates among the groups and differences in other cytokines were not identified by immunocytochemistry. Protection from clinical EAE in LR and co-immunized mice was partially abolished by anti-TGF-beta antibody treatment. Thus, protection from clinical disease following immunization with the analog LR is associated with infiltration into the CNS of a T cell population that could potentially recognize the native PLP peptide and with enhanced TGF-beta production by cells within CNS inflammatory foci.
... APLs have been extremely effective in the treatment of several animal models of human autoimmune diseases [26,33,34,35,36]. However, there are a few important differences between these animal models and their counterparts in humans: ...
... We believe that together these data suggest that if one relies on TCR-or MHC-mediated effects of APL therapy (i.e., via MHC blockade [54], TCR antagonism, partial agonism [34], or anergy [36]), it is not possible to use a single APL interindividually (across the species or individuals with different MHC backgrounds), and that even intraindividually the TCR diversity may preclude a consistent therapeutic response [35]. ...
... Experimental evidence suggests that these immunoregulatory T cells migrate into the inflammatory site and, upon crossstimulation with the native antigen that is released during tissue destruction, are activated and produce regulatory cytokines such as IL-4, IL-10, IL-13, and TGF-β [56]. The release of these anti-inflammatory cytokines subsequently down-modulates the ongoing inflammatory process in an antigen-nonspecific manner, by the above bystander suppression [35,55,56]. For example, Santambrogio et al. [35] have clearly demonstrated also in the PLP 138-151 mediated EAE in SJL/J mice that immunization with the APL with substitutions in two presumed TCR contact sites (W144→L/H147→R) protects animals coimmunized with the native encephalitogen PLP 139-151 from clinical development of EAE and its subsequent relapses. ...
An important subgroup of human cellular receptors uses peptides as signaling molecules. Modifications of these signaling peptides, usually by amino acid substitutions in crucial receptor contact sites (i.e., altered peptide ligands, APLs), is an approach for highly selective and specific modulation of the receptor function. One of the major applications of APLs is immunology, where APLs have been examined for therapeutic modulation of T cell function, both for diseases characterized by unwanted activation of T cells (e.g., autoimmune diseases) and for disorders with suboptimal T cell activation (e.g., immunotherapy of cancers and infectious disorders). APLs also occur in vivo, for example, as escape mutants of infectious agents, and play an important role in thymic positive selection. We summarize current knowledge of the basic mechanisms of the effects of APLs with special focus on T cell receptor signaling and the use of APLs for the treatment of autoimmune diseases.
... After addition of antigenic peptide PLP[139][140][141][142][143][144][145][146][147][148][149][150][151]to the culture medium, the level of empty cell-surface MHC decreased (Fig. 4A), with a concomitant increase in level of the peptide-loaded form as detected with the complexspecific antibody Y3P (Fig. 4B). Nonbinding control peptide PLP[35][36][37][38][39][40][41][42][43][44][45][46][47][48][49][50]had no effect on KL-304 or Y3P surface expression (not shown). Confocal microscopy of splenic DC confirmed the developmental regulation of empty MHC class II expression. ...
... Cells were treated with sodium azide (12 mM) to prevent endocytosis and other energy-requiring processes, washed, and then incubated with peptide in a 2-hr binding assay. Immature DC efficiently bound added antigenic peptide PLP[139][140][141][142][143][144][145][146][147][148][149][150][151], whereas control peptide PLP[35][36][37][38][39][40][41][42][43][44][45][46][47][48][49][50]did not bind, as shown by flow cytometry (Fig. 5A). Cell-surface peptide binding varied with DC developmental state, with more immature DC exhibiting more peptide-binding activity (Fig. 5A–C). ...
A monoclonal antibody specific for the empty conformation of class II MHC molecules revealed the presence of abundant empty molecules on the surface of spleen- and bone marrow-derived dendritic cells (DC) among various types of antigen-presenting cells. The empty class II MHC molecules are developmentally regulated and expressed predominantly on immature DC. They can capture peptide antigens directly from the extracellular medium and present bound peptides to antigen-specific T lymphocytes. The ability of the empty cell-surface class II MHC proteins to bind peptides and present them to T cells without intracellular processing can serve to extend the spectrum of antigens able to be presented by DC, consistent with their role as sentinels in the immune system.
... T Cell Lines and Clones. The T cell lines SP/2, SP/3, PLP/a, and PLP/c were generated and maintained as described previously (29). In brief, SJL mice were immunized subcutaneously with PLP139-151(C140S) peptide emulsified in CFA. ...
... T cell hybridomas hPLP/1, hPLP/a9.4, and hPLP/c4 were generated by a polyethylene glycol fusion of the T cell lines PLP/a and PLP/c, respec-tively, with TCR-␣ /  Ϫ BW1100 thymoma cells, selected in the presence of histone acetyltransferase (HAT) and cloned by limiting dilution as described previously (29). ...
T cell epitope peptides derived from proteolipid protein (PLP139-151) or myelin basic protein (MBP86-100) induce experimental autoimmune encephalomyelitis (EAE) in "susceptible" strains of mice (e.g., SJL/J). In this study, we show that the encephalitogenic effect of these epitopes when injected subcutaneously in complete Freund's adjuvant was significantly enhanced if administered to the animal in a multimerized form as a T cell epitope oligomer (i.e., as multiple repeats of the peptide epitope, such as 16-mers). Oligomer-treated SJL/J mice developed EAE faster and showed a more severe progression of the disease than animals treated with peptide alone. In addition, haplotype-matched B10.S mice, "resistant" to EAE induction by peptide, on injection of 16-mers developed a severe form of EAE. Even more striking, however, was the dramatic suppression of incidence and severity of the disease, seen after single intravenous injections of only 50 microg of the PLP139-151 16-mer, administered to SJL/J mice 7 d after the induction of the disease. Although relapse occurred at about day 45, an additional injection several days before that maintained the suppression. Importantly, the specific suppressive effect of oligomer treatment was also evident if EAE was induced with spinal cord homogenate instead of the single peptide antigen. By contrast, the PLP139-151 peptide accelerated rather than retarded the progression of disease.
... Substitutions of a single amino acid residue in the PLP139-151 peptide ( Table 1) to make APLs allowed for the discovery of critical residues for binding to MHC-II and TCR using MHC and TCR binding assays, respectively. 81 Because of their potential to modulate immune responses, APLs from the PLP peptide were developed as potential therapeutics to treat MS. 101 Residues W144 and H147 in PLP139-151 were respectively replaced with L144 and R147 to make the LR-APL (Table 1) with increased TCR binding properties. 81 The efficacy of LR-APL was compared with the parent PLP139-151 in suppressing EAE in mice; the result showed T cells activated by LR-APL accessed the CNS and had different antigen recognition than those of activated by the native PLP139-151 peptide. ...
The immune system defends our body by fighting infection from pathogens utilizing both the innate and adaptive immune responses. The innate immune response is generated rapidly as the first line of defense. It is followed by the adaptive immune response that selectively targets infected cells. The adaptive immune response is generated more slowly, but selectively, by targeting a wide range of foreign particles (i.e., viruses or bacteria) or molecules that enter the body, known as antigens. Autoimmune diseases are the results of immune system glitches, where the body’s adaptive system recognizes self-antigens as foreign. Thus, the host immune system attacks the self-tissues or organs with a high level of inflammation and causes debilitation in patients. Many current treatments for autoimmune diseases (i.e., multiple sclerosis (MS), rheumatoid arthritis (RA)) have been effective but lead to adverse side effects due to general immune system suppression, which makes patients vulnerable to opportunistic infections. To counter these negative effects, many different avenues of antigen specific treatments are being developed to selectively target the autoreactive immune cells for a specific self-antigen or set of self-antigens while not compromising the general immune system. These approaches include soluble antigenic peptides, bifunctional peptide inhibitors (BPI) including IDAC and Fc-BPI, polymer conjugates, and peptide-drug conjugates. Here, various antigen-specific methods of potential treatments, their efficacy, and limitations will be discussed along with the potential mechanisms of action.
... If the previous era was dedicated to the role of antibodies in disease for which Tonegawa received the Nobel Prize in 1987 (Table 2) [74], this era was that of T cells in neuroimmunology and the recognition of the importance of innate immunity (Figure 3). Following Doherty and Zinkernagel's discovery in 1974 for which they were awarded the Nobel Prize in 1996, major steps were made in identifying the T cell receptor (1983)(1984)(1985)(1986)(1987) [75,76] (Table 2), classification of T cells (1986) [77], the role of MHC peptide complex in triggering T cell responses (1991) [78], and how T cells are regulated (1995) [79] or modified using altered peptide ligands (1998) [80]. Models also made use of the emerging field of transgenic mice designed to express human proteins such as human leukocyte antigens (HLA), T cells expressing specific T-cell receptors (TCRs), markers such as green fluorescent protein (GFP) to allow tracking of cells, or generated to lack specific molecules (knock-out or deficient mice). ...
Neuroimmunology as a separate discipline has its roots in the fields of neurology, neuroscience and immunology. Early studies of the brain by Golgi and Cajal, the detailed clinical and neuropathology studies of Charcot, and Thompson's seminal paper on graft acceptance in the central nervous system kindled a now rapidly expanding research area with the aim of understanding pathological mechanisms of inflammatory components of neurological disorders. Whilst neuroimmunologists originally focussed on classical neuroinflammatory disorders such as multiple sclerosis and infections, there is strong evidence to suggest that the immune response contributes to genetic white matter disorders, epilepsy, neurodegenerative diseases, neuropsychiatric disorders, peripheral nervous system and neuro‐oncological conditions, as well as ageing. Technological advances have greatly aided our knowledge of how the immune system influences the nervous systems during development and ageing, and how such responses contribute to disease as well as regeneration and repair.
Here, we highlight historical aspects and milestones in the field of neuroimmunology and discuss the paradigm shifts that have helped provide novel insights into disease mechanisms. We propose future perspectives including molecular biological studies and experimental models that may have the potential to push many areas of neuroimmunology. Such an understanding of neuroimmunology will open up new avenues for therapeutic approaches to manipulate neuroinflammation.
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... Scale bar = 100 μm. duration in EAE (Tian et al., 2013a;Gaur et al., 1997;Nicholson et al., 1995;Fischer et al., 2000;Santambrogio et al., 1998;Ruiz et al., 1999;Karin et al., 1994;Brocke et al., 1996). ...
Neuropathic pain is a debilitating condition inmultiple sclerosis and experimental autoimmune encephalomyelitis
(EAE). Specificmyelin basic protein (MBP) peptides are encephalitogenic, andmyelin-derived altered peptide
ligands (APLs) are capable of preventing and ameliorating EAE. We investigated the effects of active
immunisation with a weakly encephalitogenic epitope of MBP (MBP87–99) and its mutant APL (Cyclo-87–
99[A91,A96]MBP87–99) on pain hypersensitivity and neuroinflammation in Lewis rats. MBP-treated rats exhibited
significant mechanical and thermal pain hypersensitivity associated with infiltration of T cells, MHC class II
expression and microglia activation in the spinal cord, without developing clinical signs of paralysis.
Co-immunisation with APL significantly decreased pain hypersensitivity and neuroinflammation emphasising
the important role of neuroimmune crosstalk in neuropathic pain.
... Należy też dodać, że weksperymentach na zwierzętach zmiana w peptydzie antygenicznym (białko proteolipidowe mieliny) jednego peptydu na inny (np. tryptofanu na glutaminę) znamiennie wpływała na różnicowanie komórek T, zwiększając wytwarzanie limfocytów Th 2 (IL-4 i IL-lO) i Th o (INF-y i IL-lO), a nie limfocytów Th 1 , co miało wpływ na zahamowanie rozwoju doświadczalnego autoimmunologicznego zapalenia mózgu i rdzenia kręgowego (19)(20)(21)(22). ...
... Scale bar = 100 μm. duration in EAE (Tian et al., 2013a;Gaur et al., 1997;Nicholson et al., 1995;Fischer et al., 2000;Santambrogio et al., 1998;Ruiz et al., 1999;Karin et al., 1994;Brocke et al., 1996). ...
Neuropathic pain is a debilitating condition inmultiple sclerosis and experimental autoimmune encephalomyelitis
(EAE). Specificmyelin basic protein (MBP) peptides are encephalitogenic, andmyelin-derived altered peptide
ligands (APLs) are capable of preventing and ameliorating EAE. We investigated the effects of active
immunisation with a weakly encephalitogenic epitope of MBP (MBP87–99) and its mutant APL (Cyclo-87–
99[A91,A96]MBP87–99) on pain hypersensitivity and neuroinflammation in Lewis rats. MBP-treated rats exhibited
significant mechanical and thermal pain hypersensitivity associated with infiltration of T cells, MHC class II
expression and microglia activation in the spinal cord, without developing clinical signs of paralysis.
Co-immunisation with APL significantly decreased pain hypersensitivity and neuroinflammation emphasising
the important role of neuroimmune crosstalk in neuropathic pain.
... No other changes were observed during the course of the experiment. 28, 31, 43, 44). We also demonstrated that EAE severity is dependent upon MBP dosage and gender. ...
Neuropathic pain is a chronic symptom of multiple sclerosis (MS) and affects nearly half of all MS sufferers. A key instigator of this pain is the pro-inflammatory response in MS. We investigated the behavioral effects of immunization with a mutant peptide of myelin basic protein (MBP), termed altered peptide ligand (APL), known to initiate immune deviation from a pro-inflammatory state to an anti-inflammatory response in experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Male and female Lewis rats were injected with vehicle control or with varying doses of 50 or 100 μg guinea pig MBP in combination with or without APL. APL-treated animals established significantly lower disease severity compared to encephalitogenic MBP-treated animals. Animals with EAE developed mechanical, but not thermal pain hypersensitivity. Mechanical pain sensitivities were either improved or normalized during periods of clinical disease in male and female APL-treated animals as compared to the encephalitogenic group. No significant changes to thermal latency were observed upon co-immunization with APL. Together these data indicate that APL ameliorates disease states and selectively mediates an analgesic effect on EAE animals.
... The immunotherapeutic effects of altered antigen peptide on autoimmune diseases have been extensively studied in recent years (24), including experimental autoimmune encephalomyelitis (25,26) and type 1 diabetes (27). Previous studies from our laboratory and others have shown that altered CII peptides prevent the development of CIA by regulating the differentiation of T helper cell subsets (28)(29)(30)(31)(32). HA 308-317 peptide is a high-affinity HLA-DR4/1-binding peptide. ...
Objective:
To investigate the inhibitory effect and possible mechanism of a novel influenza virus hemagglutinin 308-317 peptide (altered HA308-317 peptide) in collagen-induced arthritis (CIA).
Methods:
CIA was induced in DBA/1 mice by immunization with type II collagen (CII). Altered HA308-317 peptide, wild HA308-317 peptide, wild CII263-272 peptide, and irrelevant peptide were administered intranasally beginning at arthritis onset. Clinical and histologic scores were assessed, and cytokine levels were determined in the serum or in supernatants from splenocytes. Characteristics of T cell subsets in response to different peptides were analyzed both in vivo and in vitro.
Results:
Intranasal administration of wild CII263-272 peptide, wild HA308-317 peptide, or altered HA308-317 peptide could significantly ameliorate CIA, but altered HA308-317 peptide showed greater therapeutic effects than wild CII263-272 peptide and wild HA308-317 peptide. The effect of altered HA308-317 peptide was associated with a substantial decrease in production of interleukin-17 (IL-17) and interferon-γ (IFNγ) and with a marked increase in production of IL-10 and transforming growth factor β, both in serum and in supernatants from splenocytes treated with altered HA308-317 peptide. Both the number and function of CD4+ Treg cells were significantly up-regulated by altered HA308-317 peptide, with a decreased induction of Th1 cells (CD4+IFNγ+) and Th17 cells (CD4+IL-17+). Adoptive transfer of CD4+CD25+ T cells from altered HA308-317 peptide-treated mice resulted in greater suppressive capacity in ameliorating CIA severity than did adoptive transfer of CD4+CD25+ T cells from wild HA308-317 peptide-treated, wild CII263-272 peptide-treated, or irrelevant peptide-treated mice.
Conclusion:
Intranasal administration of altered HA308-317 peptide potently suppressed the severity of CIA by increasing the number and function of CD4+ Treg cells, suggesting that altered HA308-317 peptide might be a promising candidate for treatment of rheumatoid arthritis.
... 6 By crystallographic analysis and alanine scanning, it has been showed that 308Y, 311Q, 313T, 314L and 316L of HA306-318 are HLA-DR1/4 contact residues, while 309V, 310K, 312N and 315K are mainly responsible for binding with TCR. Since there has been an increase in interest in altered peptides which can be used as antigen-specific therapeutic agents in autoimmune diseases, [7][8][9][10][11] we designed a series of altered HA308-317 peptides with amino acid substitutions at TCR contact sites (YAKQATLALA) in previous in vitro studies. The results showed that these altered peptides inhibited T-cell proliferation stimulated by wild-type HA306-318 peptide or CII263-272 peptide in peripheral lymphocytes of RAand CII-specific T-cell clones. ...
There has been an increase in interest in the use of altered peptides as antigen-specific therapeutic agents in autoimmune diseases. Here we investigated the inhibitory effect and possible mechanism of an altered influenza virus haemagglutinin (HA)-derived peptide in collagen-induced arthritis (CIA). CIA was induced in DBA/1 mice by immunisation with type II collagen (CII). Altered HA308-317, wild-type HA308-317 or irrelevant peptide was administered intranasally beginning from arthritis onset. Clinical and histological scores were assessed, and cytokine levels in the serum or supernatants from splenocytes were determined. The percentages of Th1 and Th2 cells in response to different peptides were analysed by FACS both in vivo and in vitro. Our results showed that intranasal administration of altered HA308-317 peptide significantly ameliorated CIA. The therapeutic effect of altered HA308-317 peptide was associated with a substantial decrease in production of interferon (IFN)-γ, interleukin (IL)-6, monocyte chemoattractant protein (MCP)-1, anti-CII IgG, IgG1 and IgG2a antibodies, and an markedly increase in production of IL-10 and IL-4 in serum or supernatants from splenocytes treated with altered HA308-317 peptide. The percentage of Th2 (CD4(+)IL-4(+)) cells was upregulated significantly by altered HA308-317 peptide with a decreased percentage of Th1 (T helper 1; CD4(+)INF-γ(+)) cells both in vivo and in vitro. These findings suggest that altered HA308-317 peptide might be a promising candidate for rheumatoid arthritis (RA) treatment.
... The endogenous TGF-1 production is up-regulated in the CNS and presumably plays a down-modulatory role during the recovery phase of acute EAE (43,44). Although anti-TGF-1 Ab treatment in vivo aggravates EAE severity (43,45,(52)(53)(54), TGF-1 treatment can prevent EAE and suppress disease already established (42,55). Weiner et al. (45) showed that the protective effect of oral administration of myelin Ags is abrogated by anti-TGF-1. ...
Current pathogenic concepts of inflammatory demyelinating disorders such as multiple sclerosis (MS) are based on the hypothesis that a T cell-mediated autoimmune response is involved in the disease process. One of the primary goals in the in the development of immunotherapies for autoimmune diseases has been to achieve inactivation of disease-inducing lymphocytes either by direct inhibition or suppression through regulatory cells and/or cytokines. The CD26 antigen is identical with the cell surface ectopeptidase dipeptidyl peptidase IV (DP IV, EC 3.4.14.5) which is involved in regulating T cell activation and growth. Activated T cells, including those specific for myelin antigens, express high levels of CD26/DP IV. In vitro, reversible DP IV inhibitors suppress T cell proliferation and pro-inflammatory cytokine production in response to myelin antigens. Further studies will evaluate the role of DP IV inhibition in T cell-mediated inflammatory disease of the central nervous system.
... The endogenous TGF-1 production is up-regulated in the CNS and presumably plays a down-modulatory role during the recovery phase of acute EAE (43,44). Although anti-TGF-1 Ab treatment in vivo aggravates EAE severity (43,45,(52)(53)(54), TGF-1 treatment can prevent EAE and suppress disease already established (42,55). Weiner et al. (45) showed that the protective effect of oral administration of myelin Ags is abrogated by anti-TGF-1. ...
CD26 or dipeptidyl peptidase IV (DP IV) is expressed on various cell types, including T cells. Although T cells can receive activating signals via CD26, the physiological role of CD26/DP IV is largely unknown. We used the reversible DP IV inhibitor Lys[Z(NO(2))]-pyrrolidide (I40) to dissect the role of DP IV in experimental autoimmune encephalomyelitis (EAE) and to explore the therapeutic potential of DP IV inhibition for autoimmunity. I40 administration in vivo decreased and delayed clinical and neuropathological signs of adoptive transfer EAE. I40 blocked DP IV activity in vivo and increased the secretion of the immunosuppressive cytokine TGF-beta1 in spinal cord tissue and plasma during acute EAE. In vitro, while suppressing autoreactive T cell proliferation and TNF-alpha production, I40 consistently up-regulated TGF-beta1 secretion. A neutralizing anti-TGF-beta1 Ab blocked the inhibitory effect of I40 on T cell proliferation to myelin Ag. DP IV inhibition in vivo was not generally immunosuppressive, neither eliminating encephalitogenic T cells nor inhibiting T cell priming. These data suggest that DP IV inhibition represents a novel and specific therapeutic approach protecting from autoimmune disease by a mechanism that includes an active TGF-beta1-mediated antiinflammatory effect at the site of pathology.
... advances in tolerogen design have led to the development of analogues Ž . or altered peptide ligands APL in which one or more amino acids have been substituted in the native amino acid Ž sequence of an immunogenic epitope Smilek et al., 1991;Wauben et al., 1994;Samson and Smilek, 1995;Nicholson et al., 1995Nicholson et al., , 1997Gaur et al., 1997;Santambrogio et al., . 1998;Anderton et al., 1999 . Some of these strategies have been shown to induce Abystander suppressionB in which a T cell response to one epitope is suppressed by treatment with another epitope on the same or even on another Ž molecule Nicholson et al., 1995Nicholson et al., , 1997Falcone and . Bloom, 1997 . If protection with palmitoyl-pepti ...
Treatment of SJL mice either before or after challenge with palmitoylated PLP139-151 (PAL139-151) completely suppressed or considerably reduced both acute and relapsing stages of EAE induced with PLP139-151. In the presence of Pertussis toxin, treatment with PAL139-151 was less effective, but treatment with a mixture of PAL139-151 and PAL178-191, the palmitoylated PLP epitope to which T cell recognition spreads, resulted in almost complete protection. Proliferation of lymphocytes from treated mice were sharply reduced, and adoptive transfer of lymph node lymphocytes from treated mice to naive recipients resulted in the reduction of the acute phase of EAE and in delayed relapses following challenge. The results suggest that treatment with PAL139-151 leads to both anergy and the generation of regulatory cells.
... 41), and in the virus-encoded MBP peptide this residue is not acetylated. The recombinant virus presents an altered peptide ligand to the mouse, resulting in protection from autoimmune disease [42][43][44] . It should be noted that another possible explanation is that T cells specific for the cross-reacting epitope were inappropriately stimulated during infection leading to anergy or unresponsiveness owing to activationinduced cell death 45 . ...
Some viruses have the ability to modulate the development of autoimmune diseases. Virus infections have long been associated with the exacerbation of autoimmune disease, however, there is also evidence that viruses can actually protect against autoimmune disease. Several experimental models have been developed to investigate how some virus infections can prime for and trigger autoimmunity whereas others ameliorate the pathway leading to clinical disease. It is possible that the type I interferons, via interleukin 12, provide the link between viruses and autoimmunity.
... Furthermore, the 1B6 transgenic mice do not develop disease even after immunization with the PLP 139-151 peptide, although inflammatory lesions can be seen in the brains of immunized mice. The appearance of lesions in the CNS of clinically normal mice following immunization with PLP 139-151 is consistent with data from mice protected with altered peptides that developed CNS lesions but were resistant to clinical disease (74,80); this argues for an active process by which this repertoire may lead to protection. ...
T cells that can respond to self-antigens are present in the peripheral immune repertoire of all healthy individuals. Recently we have found that unmanipulated SJL mice that are highly susceptible to EAE also maintain a very high frequency of T cells responding to an encephalitogenic epitope of a myelin antigen proteolipid protein (PLP) 139-151 in the peripheral repertoire. This is not due to lack of expression of myelin antigens in the thymus resulting in escape of PLP 139-151 reactive cells from central tolerance, but is due to expression of a splice variant of PLP named DM20, which lacks the residues 116-150. In spite of this high frequency, the PLP 139-151 reactive cells remain undifferentiated in the periphery and do not induce spontaneous EAE. In contrast, SJL TCR transgenic mice expressing a receptor derived from a pathogenic T cell clone do develop spontaneous disease. This may be because in normal mice, autoreactive cells are kept in check by an alternate PLP 139-151 reactive nonpathogenic repertoire, which maintains a balance that keeps them healthy. If this is the case, selective activation of one repertoire or the other may alter susceptibility to autoimmune disease. Since T cells are generally cross-reactive, besides responding to nonself-antigens, they also maintain significant responses to self-antigens. Based on the PLP 139-151 system, we propose a model in which activation with foreign antigens can result in the generation of pathogenic memory T cells that mediate autoimmunity. We also outline circumstances under which activation of self-reactive T cells with foreign antigens can generate selective tolerance and thus generate protective/regulatory memory against self while still maintaining significant responses against foreign antigens. This provides a mechanism by which the fidelity and specificity of the immune system against foreign antigens is improved without increasing the potential for developing an autoimmune disease.
... Both IL-10 and TGFh, produced by the MOG APL selected for therapeutic use in vivo in the current study, have been associated with inhibition of EAE and administration of antibodies to TGFh and IL-10 enhance EAE (Cannella et al., 1996). Antigen specific therapy in EAE is augmented with IL-10 (Slavin et al., 2001) and abrogated by antibodies to IL-10 and TGFh (Sundstedt et al., 2003) and IL-10 and TGFh expression levels are higher in the brains of animals with EAE following treatment with peptide analogues (Santambrogio et al., 1998). We also observed TGFh and IL-10 production in vitro by non-encephalitogenic K and A substituted MOG 8 – 21 peptides however, despite the selection of APL that preferentially produce TGFh and/or IL-10 in vitro, established EAE disease was most effectively inhibited with the WT MOG 8 –21 peptide coupled to splenocytes. ...
Altered peptide ligands (APL) are highly effective in inhibiting experimental autoimmune encephalomyelitis (EAE) in rodents although clinical trials in multiple sclerosis reveal severe limitations probably due to the diverse and differential effects of APL in vivo compared to in vitro. Myelin oligodendrocyte glycoprotein (MOG 8-21) induces relapsing EAE in ABH (A(g7)) mice associated with broadening of the autoimmune repertoire thus providing a dynamic system to examine the efficacy of peptide analogues. Subtle changes in MOG 8-21 dramatically influenced disease susceptibility and T cell responses in vitro. Non-encephalitogenic APL that induce production of the 'regulatory' cytokines IL-10 and/or TGFbeta and concomitant low levels of the 'proinflammatory' cytokines IFNgamma and TNFalpha modulated relapsing EAE but were far less effective than the 'proinflammatory' wild-type MOG 8-21 peptide. These data reveal that APL differ greatly in their ability to activate encephalitogenic T cells. The extensive heterogeneity of responses of APL in vitro suggests that selection of APL on this criteria is highly unpredictable and probably less effective for therapy than selecting the dominant wild-type epitope and delivering it using a tolerogenic route.
... The serum IFN-in rats treated with altered CII263-272 peptide was lower than that in rats treated with wild CII263-272 peptide (*P < 0.05). No diVerence was found in the levels of models of human autoimmune diseases151617. RA is a systemic autoimmune disease mediated by T cells. The complex of antigenic peptide, HLA-DR1/4 and T cell is the primary signal of T cell activation [18]. ...
Rheumatoid arthritis (RA) is a systemic autoimmune disease mediated by T cells. Collagen type II (CII) is one of the autoantigens associated with RA. CII263-272 is a predominant CII antigenic peptide that can induce T-cell activation upon binding to MHC and interaction with the appropriate T-cell receptor (TCR). Altered CII263-272 peptides with substitution of specific amino acids could bind to RA-associated HLA-DR4/1 with no T cell stimulating effects and could inhibit T cell activation in RA. We performed this study to evaluate the effect of mucosal administration and to explore the mechanism of the inhibitory effect of altered CII263-272 peptide (267Q-->A, 270K-->A and 271G-->A) on collagen induced arthritis (CIA). CIA was induced in Lewis rats by immunization with bovine CII. Altered CII263-272 peptide was given intranasally beginning from arthritis onset. Wild CII263-272 peptide or PBS was administered as controls. Therapeutic effects were evaluated by arthritis scores, body weight change, and joint pathologic scores. The anti-CII antibody and its subtypes and the cytokines, IFN-gamma, IL-10, and IL-17 were measured with ELISA. Foxp3+CD4+CD25+ regulatory T cell induction was assessed by FACS analysis. Following treatment with the altered CII263-272 peptide, arthiritis scores were reduced and body weight was increased. The altered CII263-272 peptide could retard the histologic lesion of the joints. The titers of anti-CII antibodies IgG2a in altered CII263-272 peptide treated rats decreased markedly compared to PBS-treated rats. The serum levels of IFN-gamma in rats treated with altered peptide was lower than that of rats treated with wild CII263-272 peptide and PBS. No differences were observed in the levels of serum IL-10 among the three groups. The altered CII263-272 peptide could decrease serum level of IL-17 and increase peripheral Foxp3+CD4+CD25+ T cells at early stage of CIA. Mucosal administration of altered CII263-272 peptide could effectively inhibit the progression of CIA. Altered CII263-272 peptide could suppress Th17 cells and expand regulatory T cells in the early stage of the disease. The IgG2a subtype of anti-CII antibodies and IFN-gamma were reduced and in vivo Th1 responses were inhibited as a result of altered CII peptide treatment. Altered CII peptide is likely therapeutic in RA.
Antigen-specific therapy for multiple sclerosis may lead to a more effective therapy by induction of tolerance to a wide range of myelin-derived antigens without hampering the normal surveillance and effector function of the immune system. Numerous attempts to restore tolerance toward myelin-derived antigens have been made over the past decades, both in animal models of multiple sclerosis and in clinical trials for multiple sclerosis patients. In this review, we will give an overview of the current approaches for antigen-specific therapy that are in clinical development for multiple sclerosis as well provide an insight into the challenges for future antigen-specific treatment strategies for multiple sclerosis.
T cell epitope peptides derived from proteolipid protein (PLP130-15l) or myelin bas ic protein (MBP86-100) induce experimental autoimmune encephalomyelitis (EAE) in “susceptible” strains of mice (e.g., SJL/J). In this study, we show that the encephalitogenic effect of these epitopes when injected subcutaneously in complete Freund’s adjuvant was significantly enhanced if administered to the animal in a multimerized form as a T cell epitope oligomer (i.e., as multiple repeats of the epitope, such as 16-mers). Oligomer-treated SJL/J mice developed EAE faster and showed a more severe progression of the disease than animals treated with peptide alone. In addition, haplotype-matched B10. S mice, “resistant” to EAE induction by peptide, on injection of 16-mers developed a severe form of EAE. Even more striking, however, was the dramatic suppression of incidence and severity of the disease, seen after single intravenous injections of only 50μg of the PLP139-151 16-mer, administered to SJL/J mice 7 d after the induction of the disease. Although relapse occurred at about day 45, an additional injection several days before that maintained the suppression. Importantly, the specific suppressive effect of oligomer treatment was also evident if EAE was induced with spinal cord homogenate instead of the single peptide antigen. By contrast, the PLP139-151 peptide accelerated rather than retarded the progression of disease.
Multiple sclerosis (MS) is a chronic inflammatory/demyelinating disease of the central nervous system (CNS) with various degrees of axonal loss [1, 2] in which autoreactive CD4+ T cells are considered to play a crucial role [3–5]. In different forms of experimental autoimmune encephalomyelitis (EAE), which serves as an animal model of MS, a number of myelin, glial components, and even heat-shock antigens can serve as targets for the encephalitogenic process [6–8]. In order to identify similar target antigens in humans, human myelin-specific T cells have been studied in great detail during the last decade. Many parallels have been found that are shared by encephalitogenic cells in EAE and T cells isolated from MS patients, i.e., they are CD4+ T-helper cells that frequently express a T-helper 1 (Th1)-proinflammatory phenotype with high secretion of interferon (IFN)-γ and tumor necrosis factor (TNF)-α/β [9–11]. Furthermore, they often recognize epitopes similar to those identified as encephalitogenic in animals and frequently are restricted by MS-associated HLA-DR molecules such as HLA-DRB 1*1501 or -DRB5*0101 [12–15]. However, such autoreactive T cells have also been isolated from healthy donors [12, 16–19], which seemed to contradict their pathophysiological relevance in MS.
Neuropathic pain is a debilitating condition in multiple sclerosis and experimental autoimmune encephalomyelitis (EAE). Specific myelin basic protein (MBP) peptides are encephalitogenic, and myelin-derived altered peptide ligands (APLs) are capable of preventing and ameliorating EAE. We investigated the effects of active immunisation with a weakly encephalitogenic epitope of MBP (MBP87-99) and its mutant APL (Cyclo-87-99[A(91),A(96)]MBP87-99) on pain hypersensitivity and neuroinflammation in Lewis rats. MBP-treated rats exhibited significant mechanical and thermal pain hypersensitivity associated with infiltration of T cells, MHC class II expression and microglia activation in the spinal cord, without developing clinical signs of paralysis. Co-immunisation with APL significantly decreased pain hypersensitivity and neuroinflammation emphasising the important role of neuroimmune crosstalk in neuropathic pain.
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Current treatments for autoimmune diseases are typically non-specific anti-inflammatory agents that affect not only the autoreactive cells but also the parts of the immune system that are required to maintain health. There is a need for the development of antigen-specific therapeutic agents that can effectively prevent the autoimmune attack while leaving the rest of the immune system functioning as normal. The simplest way to achieve this is using the autoantigen itself as a tolerizing agent; however, there is some risk involved with administering a potentially pathogenic antigen. In this review, we focus instead on the development and use of modified T cell receptor (TCR) ligands, in which the peptide ligand is modified to change the response by the T cell from a disease inducing to a protective response, and still retain the antigen-specificity necessary to target the autoreactive T cells. We review the use of modified TCR ligands as therapeutic agents in animal models of autoimmunity and in human autoimmune disease, and finally consider how they need to be improved in order to use them effectively in patients with autoimmune disease.
CD4 + T-cells play a key role in the pathogenesis of multiple sclerosis (MS). Altered peptide ligands capable of modulating T-cell autoreactivity are considered a promising strategy for development of antigen-specific therapies for MS. Since peptides are inherently unstable, the current study explored single β-amino acid substitution as a means of stabilizing an epitope of myelin oligodendrocyte glycoprotein. β-amino acid substitution at position 44, the major T-cell receptor contact residue, increased the half-life of active metabolites. Vaccination with one altered peptide, MOG44βF, conferred protection from EAE, decreased T-cell autoreactivity and pro-inflammatory cytokine production. Additional studies using MOG44βF in an oral treatment regimen, administered after EAE induction, also attenuated disease severity. Thus, altered peptides such as those reported here may lead to the development of novel and more specific treatments for MS.
Experimental autoimmune encephalomyelitis (EAE) is an animal model for human multiple sclerosis (MS). The disease is initiated
by myelin-specific T cells, but mediated by cells of both the adaptive and innate immune system. Therefore, strategies targeting
either lymphocytes or myeloid cells can be effective in modulating EAE. In this chapter, we will review some of the strategies
that have been developed to treat or prevent EAE. Our emphasis will be on those strategies that are still in the developmental
stage, but may have the potential to be used in the clinic in the future
The importance of transforming growth factor-beta-1 (TGF-β1) in immunoregulation and tolerance has been increasingly recognized. It is now proposed that there are populations of regulatory T cells (T-reg), some designated T-helper type 3 (Th3), that exert their action primarily by secreting this cytokine. Here, we emphasize the following concepts: (1) TGF-β1 has multiple suppressive actions on T cells, B cells, macrophages, and other cells, and increased TGF-β1 production correlates with protection and/or recovery from autoimmune diseases; (2) TGF-β1 and CTLA-4 are molecules that work together to terminate immune responses; (3) Th0, Th1 and Th2 clones can all secrete TGF-β1 upon cross-linking of CTLA-4 (the functional significance of this in autoimmune diseases has not been reported, but TGF-β1-producing regulatory T-cell clones can produce type 1 inflammatory cytokines); (4) TGF-β1 may play a role in the passage from effector to memory T cells; (5) TGF-β1 acts with some other inhibitory molecules to maintain a state of tolerance, which is most evident in immunologically privileged sites, but may also be important in other organs; (6) TGF-β1 is produced by many cell types, is always present in the plasma (in its latent form) and permeates all organs, binding to matrix components and creating a reservoir of this immunosuppressive molecule; and (7) TGF-β1 downregulates adhesion molecules and inhibits adhesion of leukocytes to endothelial cells. We propose that rather than being passive targets of autoimmunity, tissues and organs actively suppress autoreactive lymphocytes. We review the beneficial effects of administering TGF-β1 in several autoimmune diseases, and show that it can be effectively administered by a somatic gene therapy approach, which results in depressed inflammatory cytokine production and increased endogenous regulatory cytokine production.
Multiple sclerosis (MS) is an inflammatory demyelinating disease affecting the central nervous system (CNS) and a frequent cause of neurological disability in young adults. Multifocal inflammatory lesions in the CNS white matter, demyelination, oligodendrocyte loss, axonal damage, as well as astrogliosis represent the histological hallmarks of the disease. These pathological features of MS can be mimicked, at least in part, using animal models. This review discusses the current concepts of the immune effector mechanisms driving CNS demyelination in murine models. It highlights the fundamental contribution of transgenesis in identifying the mediators and mechanisms involved in the pathophysiology of MS models.
This review is a personal memoir of the history of proteolipids and is limited to aspects of the field with which the author has been involved in one way or another. The discovery of proteolipids was a serendipitous observation made in the course of the study of sulfatides. Initial focus was on the chemical characterization of brain proteolipids, their behavior under different conditions and their identification as the major protein of CNS myelin. The sequence of PLP was obtained using solid phase protein sequencing techniques. This, in turn, made possible a new era in which biochemical, cellular and molecular approaches could be applied to address new questions about PLP. Identification of genetic defects in the PLP molecule and its regulation has contributed to understanding myelin biology. Studies of the encephalitogenic activity of PLP in animal models have influenced the views of inflammatory processes in multiple sclerosis. Despite remarkable progress, much remains to be learned about the structure and function of PLP.
Oral tolerance is a long-recognized mechanism of inducing antigen-specific peripheral immune tolerance. In the past decade a large number of investigators have successfully applied oral tolerance to the treatment of autoimmune diseases in animal models by feeding autoantigens, and human trials have begun with this approach as well (1). In this issue of the Proceedings, Teitelbaum et al. (2) report that oral tolerance using Copolymer 1 (Cop 1, Copaxone, glatiramer acetate), which simulates myelin basic protein (MBP) immunologically, is effective in both the rat and mouse models of experimental allergic encephalomyelitis (EAE) and indeed may be more effective than MBP itself. Injectible Cop 1 has been used successfully to treat both EAE (3) and the human disease multiple sclerosis (MS) (4). These results raise the possibility that orally administered Cop 1 may be an effective treatment for MS.
The term oral tolerance (OT) describes the antigen-specific suppression of immune responses following the feeding of the antigen. While some common features with other forms of induction of systemic tolerance have been disclosed, OT can be distinguished by certain immunologic characteristics. Thus, work in experimental animal models revealed the importance of intestinal antigen processing, especially antigen processing in the Peyer's patches, in inducing OT. It has become clear that suppressive T cell cytokines derived from mucosal sites play a major role in mediating OT. The variation of the dose of fed antigen and the modulation of the cytokine milieu both have influences on the underlying immunologic mechanisms active suppression, clonal anergy and clonal deletion following oral antigen uptake. In several animal models of autoimmunity the disease activity can be suppressed by feeding oral autoantigen. Based on these results, recent clinical studies have begun to explore OT as a means to treat autoimmune disorders such as multiple sclerosis, rheumatoid arthritis and diabetes.
Co-immunization with an altered peptide ligand (LR) partially protects SJL mice from proteolipid protein peptide 139-151-induced experimental allergic encephalomyelitis [Kuchroo, V.K., Greer, J.M., Kaul, D., Ishioka, G.Y., Franco, A., Sette, A., Sobel, R.A., Lees, M.B., 1994. A single TCR antagonist peptide inhibits experimental allergic encephalomyelitis mediated by a diverse T cell repertoire. J. Immunol. 153, 3326-3336; Santambrogio, L., Lees, M.B., Sobel, R.A., 1998. Altered peptide ligand modulation of experimental allergic encephalomyelitis: immune responses within the CNS. J. Neuroimmunol. 81, 1-13]. Clinical protection was noted despite extensive central nervous system inflammation observed after co-immunization with native and altered peptides. To extend our previous reports on this model, we now compare MHC class II expression and antigen presenting cell activity of cells associated with the blood-brain barrier in diseased and protected mice. Immunohistochemical studies identified MHC class II products on both the endothelial and microglial/macrophage populations. Ex vivo experiments suggested a correlation between the reduced clinical disease observed in the co-immunized mice and the antigen presenting activity of cells at the blood-brain barrier. The results suggest that antigen presenting activity is primarily mediated by macrophage-lineage cells of the central nervous system.
A combination of mutagenesis, computer modeling and immunoreactivity has been used to develop a structural model of a segment of the glutamate receptor (GluR), termed GluR3B, which is bound by receptor-activating autoantibodies. In this model, the GluR3B epitope is located in a reverse hairpin loop that places key residues important for antibody recognition and receptor activation in a linear arrangement on the solvent-exposed surface. The conformation of the loop is stabilized by a hydrophobic core which is critical for functional integrity of the epitope. The proximity of the amino- and carboxy-terminal residues suggested that the GluR3B peptide could be cyclized without diminishing immunoreactivity through replacement of these residues with cysteines and formation of a disulfide bond. This prediction was confirmed experimentally since the cyclized peptide retained full immunoreactivity. The model provides insight into GluR subunit-specific functional diversity and the role of autoantibodies to this region in neurological disease.
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.
Experimental autoimmune encephalomyelitis (EAE) is a T helper 1 (Th1) cell mediated demyelinating disease and the principal animal model for multiple sclerosis. Spinal cords from SJL mice primed with proteolipid protein peptide 139-151 (pPLP) expressed the chemokines RANTES, MCP-1, MIP-2, KC, MIP-1alpha, MIP-1beta, Mig, and fractalkine. We also identified IP-10 in these samples and described a sequence polymorphism in this transcript. Chemokine expression was specific for tissues of the central nervous system. MCP-1, IP-10, and MIP-2 RNA expression significantly correlated with clinical score. Chemokine receptor expression generally correlated with ligand expression. pPLP-primed mice expressed the Th1-associated markers CCR5 and CXCR3 on mononuclear cells. In addition, cells expressing CCR1, CCR2, CCR3, CCR4, CCR8, and CXCR2 were detected. Here we demonstrate that altered peptide ligand (APL)-induced protection from EAE was accompanied by modulation of chemokine and chemokine receptor expression. Spinal cord tissue sections from APL-protected mice showed greatly reduced levels of all chemokines and of CCR1, CCR5, CCR8, CXCR2 and CXCR3. The Th2-associated chemokine receptors CCR3 and CCR4 were found in protected mice, supporting the hypothesis that Th1 but not Th2 cells are down-regulated by APL treatment. This report concludes that chemokines and chemokine receptors can be useful tools to follow modulation of autoimmune disease.
Current therapies for autoimmune diseases and allergy involve general immune suppression. However, the ideal therapy should specifically eliminate or modulate the (auto)pathogenic immune response or, alternatively, it should reinforce the regulatory response, without affecting the overall function of the immune system. This could be achieved by antigen-specific immunotherapy. Antigen-specific immunotherapy has received ample attention in the last years, and several clinical trials attempting to treat autoimmune diseases or allergy through the induction of antigen-specific tolerance or immune deviation have been conducted, albeit with varying success. Recent advances in our understanding of peripheral tolerance, regulatory T cells, and routes of antigen administration have resulted in better insight into the different working mechanisms and potential target molecules of antigen-specific immunotherapy. The experimental animal models and new technological developments force the pace in the development of these immunotherapies. The current review addresses several aspects of antigen-specific immunotherapies and focuses on the mechanisms of the different approaches in experimental autoimmune and allergy models.
Experimental autoimmune myasthenia gravis can be induced in some strains of mice and rats by immunizing with acetylcholine receptor. Also, epidemiologic studies demonstrate an MHC linkage of myasthenia gravis in the man. In order to obtain direct experimental evidence for the influence of the genes of the MHC complex in the development of myasthenia gravis, we used mice transgenic to individual HLA molecules. We observed an increased susceptibility to the disease in HLA DQ8 transgenic mice compared to HLA DQ6 transgenic mice ( J. Immunol. 160:4169; 1998). These mice lacked endogenous mouse class II molecules. In the present study we mapped the cryptic and dominant sequences on the extra cellular region of human acetylcholine receptor. Although some epitopes (e.g., alpha11-30, alpha141-160, alpha171-190) were common between DQ8 and DQ6 transgenic mice, several others were disparately recognized. We also found a functional dichotomy in T cells from mice differing by one MHC molecule (HLA DQ8 or DQ6) when primed by sequences immunodominant in DQ8 and DQ6 tg mice. Differential disease manifestation in the two different HLA transgenic mice could be explained not only by differential recognition of peptides by these antigen presenting molecules, but also by the difference in the functional profile of T cells generated when primed by promiscuous sequence regions.
Women with multiple sclerosis have significantly diminished disease activity during pregnancy. The purpose of our study was to identify the underlying mechanism for the diminished disease activity. We found that during the period of late pregnancy there is protection against paralysis, during both the induction and effector phases of relapsing experimental autoimmune encephalomyelitis, a mouse model of multiple sclerosis. We did not find any changes in the cytokine secretion profiles or the proliferative activity of autoreactive T cells from mice induced during late pregnancy compared with virgin controls. In mice mated after disease onset, the inflammatory histologic lesions did not clear, despite marked clinical improvement during pregnancy. We found evidence for a serum factor present in late pregnancy that suppresses T cell activation. In the presence of sera taken from mice late in pregnancy, the proliferative response and IL-2 production of proteolipid protein p139-151-specific T cells were significantly diminished as compared with stimulation in the presence of normal mouse sera. In conclusion, serum from late pregnancy has the capacity to down-regulate T cell responses and might be associated with the amelioration of disease activity in experimental autoimmune encephalomyelitis.
In the inflammatory diseases autoimmune encephalomyelitis (EAE) and multiple sclerosis (MS) the occurrence, severity, course
and resolution of disease are dependent on a complex interaction of cells, cytokines, chemokines and myriad other mediators.
This chapter describes the role of what, in the authors opinions, are the most influential cytokines in these two diseases.
To understand the effect of the various cytokines it is important to bear in mind that most are highly pleiotrophic and like
most biological molecules, their effects are dose, time and site dependent. Targeting cytokines as therapy for these diseases
is a feasible approach but careful consideration must be given not only to the cytokine targeted but the stage of the disease
process being targeted. A ‘ready reckoner’ of cytokine function in relation to CNS inflammation is presented at the end of
the chapter.
The existence of T cells restricted for the MHC I-like molecule CD1 is well established, but the function of these cells is still obscure; one implication is that CD1-dependent T cells regulate autoimmunity. In this study, we investigate their role in experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis, using CD1-deficient mice on a C57BL/6 background. We show that CD1-/- mice develop a clinically more severe and chronic EAE compared with CD1+/+ C57BL/6 mice, which was histopathologically confirmed with increased demyelination and CNS infiltration in CD1-/- mice. Autoantigen rechallenge in vitro revealed similar T cell proliferation in CD+/+ and CD1-/- mice but an amplified cytokine response in CD1-/- mice as measured by both the Th1 cytokine IFN-gamma and the Th2 cytokine IL-4. Investigation of cytokine production at the site of inflammation showed a CNS influx of TGF-beta1-producing cells early in the disease in CD1+/+ mice, which was absent in the CD1-/- mice. Passive transfer of EAE using an autoreactive T cell line induced equivalent disease in both groups, which suggested additional requirements for activation of the CD1-dependent regulatory pathway(s). When immunized with CFA before T cell transfer, the CD1-/- mice again developed an augmented EAE compared with CD1+/+ mice. We suggest that CD1 exerts its function during CFA-mediated activation, regulating development of EAE both through enhancing TGF-beta1 production and through limiting autoreactive T cell activation, but not necessarily via effects on the Th1/Th2 balance.
It has been reported that collagen II (CII)-derived peptide induced T-cell activation via its amino acids responsible for T-cell receptor (TCR) recognition. In this study, three altered CII263-272 peptide ligands (APL) containing multiple substitutions of TCR contact residues were synthesized. Their roles in inhibition of T-cell activation were evaluated in peripheral blood lymphocytes (PBL) of rheumatoid arthritis (RA) in vitro. It was shown that 41% (25/61) of RA patients were responsive to the wild-type antigenic CII263-272. In contrast, marginal or silent T-cell responses to the three APLs were found, accompanied by inhibitory effects on secretion of Th1 type cytokines and expression of cell surface markers, CD69 and CD25. In addition, T-cell activation induced by the wild-type antigenic CII263-272 was inhibited by all the three APLs in a dose-dependent manner. It is demonstrated that APLs with substitutions of TCR contact residues are capable of down-regulating T-cell responses in PBLs of RA, suggesting that the CII-derived APLs are potentially therapeutic in RA.
Peptide immunotherapy is a promising alternative for treating allergic diseases. One way to enhance the efficacy of peptide immunotherapy is to use altered peptide ligands (APLs) that contain amino acid substitutions compared with the natural peptide.
To evaluate the potential of an APL of the immunodominant epitope of lipocalin allergen Bos d 2 for peptide immunotherapy.
Peripheral blood CD4(+) T-cell responses of 8 HLA-DR4-positive subjects to the natural ligand of Bos d 2 (p127-142) or to an APL (pN135D) were analyzed by MHC class II tetramer staining after in vitro expansion with the peptides. Long-term T-cell lines (TCLs) were induced with the peptides, and the cytokine production, cross-reactivity, and T-cell receptor Vbeta subtype expression of the TCLs were analyzed.
CD4(+) T cells specific for both p127-142 and pN135D were readily detected in peripheral blood after a single in vitro stimulation. Whereas the TCLs induced with p127-142 were T(H)2/T(H)0-deviated, those induced with pN135D were T(H)1/T(H)0-deviated and highly cross-reactive with p127-142. Moreover, the pN135D-induced TCLs appeared to use a broader repertoire of T-cell receptor Vbeta subtypes than those induced with p127-142.
An APL of an immunodominant allergen epitope was able to induce a novel T(H)1-deviated T-cell population cross-reactive with the natural epitope in vitro. This cell population could have a therapeutic immunomodulatory function in vivo through bystander suppression.
These results support the idea that altered peptide ligands may be used to enhance the efficacy of peptide immunotherapy.
Multiple sclerosis (MS) is an inflammatory demyelinating disease of the CNS, characterized pathologically by a perivascular infiltrate consisting predominantly of T cells and macrophages. Although its aetiology remains unknown, several lines of evidence support the hypothesis that autoimmune mechanisms play a major role in the development of the disease. Several widely used disease-modifying agents are approved for the treatment of MS. However, these agents are only partially effective and their ability to attenuate the more progressive phases of the disease is not clear at this time. Therefore, there is a need to develop improved treatment options for MS.
This article reviews the role of several novel, selective vaccine strategies that are currently under investigation, including: (i) T-cell vaccination (TCV); (ii) T-cell receptor (TCR) peptide vaccination; (iii) DNA vaccination; and (iv) altered peptide ligand (APL) vaccination.
The administration of attenuated autoreactive T cells induces regulatory networks to specifically suppress pathogenic T cells in MS, a strategy named TCV. The concept of TCV was based on the experience of vaccination against aetiological agents of infectious diseases in which individuals are purposely exposed to an attenuated microbial pathogen, which then instructs the immune system to recognize and neutralize it in its virulent form. In regard to TCV, attenuated, pathogenic T cells are similarly used to instruct the immune system to recognize and neutralize disease-inducing T cells.
In experimental allergic encephalomyelitis (EAE), an animal model for MS, pathogenic T cells use a strikingly limited number of variable-region elements (V region) to form TCR specific for defined autoantigens. Thus, vaccination with peptides directed against these TCR structures may induce immunoregulatory mechanisms, thereby preventing EAE. However, unlike EAE, myelin-reactive T cells derived from MS patients utilize a broad range of different V regions, challenging the clinical utility of this approach. Subsequently, the demonstration that injection of plasmid DNA encoding a reporter gene into skeletal muscle results in expression of the encoded proteins, as well as in the induction of immune responses in animal models of autoimmunity, was explored as another strategy to re-establish self-tolerance. This approach has promise for the treatment of MS and, therefore, warrants further investigation.
APLs are molecules in which the native encephalitogenic peptides are modified by substitution(s) of one or a few amino acids critical for contact with the TCR. Depending on the substitution(s) at the TCR contact residues of the cognate peptide, an APL can induce immune responses that can protect against or reverse EAE. However, the heterogeneity of the immune response in MS patients requires further study to determine which patients are most likely to benefit from APL therapy.
Other potential approaches for vaccines in MS include vaccination against axonal growth inhibitors associated with myelin, use of dendritic cells pulsed with specific antigens, and active vaccination against proinflammatory cytokines.
Overall, vaccines for MS represent promising approaches for the treatment of this devastating disease, as well as other autoimmune diseases.
A new method of total RNA isolation by a single extraction with an acid guanidinium thiocyanate-phenol-chloroform mixture is described. The method provides a pure preparation of undegraded RNA in high yield and can be completed within 4 h. It is particularly useful for processing large numbers of samples and for isolation of RNA from minute quantities of cells or tissue samples.
We previously described a synthetic peptide of myelin proteolipid protein (PLP), peptide 139-151, which induces experimental allergic encephalomyelitis in SJL/J (H-2s) mice. We have now identified an additional determinant, PLP residues 178-191, that is also a potent encephalitogen in this strain. When PLP peptide 178-191 was compared with peptide 139-151 on an equimolar basis, the day of onset of disease induced by PLP 178-191 was earlier, but the incidence, severity, and histologic features were indistinguishable. Lymph node cells from animals immunized with the whole PLP molecule responded to both PLP 178-191 and 139-151, suggesting immunologic codominance of the two epitopes. PLP 178-191 elicited stronger proliferative responses and this may relate to the earlier onset of disease induced with this peptide. Two CD4+, peptide-specific, I-A(s)-restricted T cell lines, selected by stimulation of lymph node cells with either PLP 178-191 or 139-151, were each encephalitogenic in naive syngeneic mice. The presence of multiple encephalitogenic codominant PLP epitopes within a single mouse strain emphasizes the complexity of the immune response to PLP and its potential as a target Ag in autoimmune demyelinating diseases.
Experimental autoimmune encephalomyelitis (EAE) is an experimental demyelinating disease of rodents. In (PL/J x SJL) F1 mice, it is induced by immunization with the myelin basic protein peptide Ac1-11. Ac1-11 [4A], a myelin basic protein peptide analog with a single amino acid substitution, (i) binds to class II major histocompatibility complex molecules and stimulates encephalitogenic T cells in vitro better than Ac1-11, (ii) is nonimmunogenic and nonencephalitogenic in vivo in (PL/J x SJL)F1 mice, (iii) prevents EAE when administered before or at the time of immunization with Ac1-11, and (iv) prevents EAE when administered later, near the time of disease onset. Initial studies suggest that Ac1-11 [4A] does not prevent EAE by competitive inhibition or by activation of regulatory cells. Thus, substitution of a single amino acid in a myelin basic protein peptide confers the capacity to prevent rather than induce EAE, even after peptide-specific encephalitogenic T cells have been activated.
Uncertainty regarding pathogenic mechanisms has been a major impediment to effective prevention and treatment for human neurologic diseases such as multiple sclerosis, tropical spastic paraparesis, and AIDS demyelinating disease. Here, we implicate lymphotoxin (LT) (tumor necrosis factor beta [TNF-beta]) and TNF-alpha in experimental allergic encephalomyelitis (EAE), a murine model of an autoimmune demyelinating disease. In this communication, we report that treatment of recipient mice with an antibody that neutralizes LT and TNF-alpha prevents transfer of clone-mediated EAE. LNC-8, a myelin basic protein-specific T cell line, produces high levels of LT and TNF-alpha after activation by concanavalin A, antibody to the CD-3 epsilon component of the T cell receptor, or myelin basic protein presented in the context of syngeneic spleen cells. LNC-8 cells transfer clinical signs of EAE. When LNC-8 recipient mice were also treated with TN3.19.12, a monoclonal antibody that neutralizes LT and TNF-alpha, the severity of the transferred EAE was reduced, while control antibodies did not alter the disease. The effect of anti-LT/TNF-alpha treatment was long lived and has been sustained for 5 mo. These findings suggest that LT and TNF-alpha and the T cells that produce them play an important role in EAE.
A new method of total RNA isolation by a single extraction with an acid guanidinium thiocyanate-phenol-chloroform mixture is described. The method provides a pure preparation of undegraded RNA in high yield and can be completed within 4 h. It is particularly useful for processing large numbers of samples and for isolation of RNA from minute quantities of cells or tissue samples.
Previously, six T cell clones, which are specific for an encephalitogenic determinant of myelin proteolipid protein (PLP) peptide residues 139 to 151 (HSLGKWLGHPDKF), were derived from SJL mice and shown to use diverse TCR genes. To design TCR antagonist peptides that could interfere with the activation of these clones in vitro and inhibit experimental allergic encephalomyelitis (EAE) in vivo, we first determined the TCR and MHC contact residues of the encephalitogenic peptide. The analysis indicated that residues 144 (tryptophan) and 147 (histidine) were the TCR binding sites and that residues 145 (leucine) and 148 (proline) were important for MHC class II (IAs) binding. On the basis of this information, a peptide analogue (leucine 144/arginine 147), in which both of the major TCR contact residues were substituted, was synthesized. This analogue acts as a TCR antagonist for the panel of PLP 139-151-specific T cell clones, does not cause EAE by itself, blocks the induction of disease by the native 139-151 peptide, and prevents clinical disease progression if administered at the first signs of disease. Thus, although multiple TCR genes are used by PLP 139-151-specific clones, a single peptide analogue can interfere with the disease process. This approach should be feasible for designing peptide analogues that can be tested for therapeutic efficacy in human autoimmune diseases in which the pathogenic Ags are known and TCR use is diverse.
An immunodominant epitope of myelin basic protein (MBP), VHFFKNIVTPRTP (p87-99), is a major target of T cells in lesions of multiple sclerosis (MS) and in experimental allergic encephalomyelitis (EAE). T cells found in EAE lesions bear the same amino acids in the third complementary determining region of the T cell receptor (TCR) as those found in MS lesions. We analyzed the trimolecular interactions between MBP p87-99, class II major histocompatibility complex (MHC), and TCR, and designed soluble inhibitors for therapy. F, N, I, and V at positions 90, 92, 93, and 94 interact with MHC, whereas K, T, and P at positions 91, 95, and 96 interact with TCR. The peptides, p87-99[95T > A] and p87-99[96P > A] could compete more effectively with p87-99 for binding to MHC and could antagonize the in vitro response to T cells to p87-99 more effectively than p87-99[91K > A]. However, only p87-99[91K > A] prevented and reversed EAE, indicating that the extent of MHC or TCR competition does not predict success in treating EAE. To elucidate the mechanism of inhibition of EAE, draining lymph node cells from rats immunized with the native peptide alone or together with each of the three TCR antagonists were challenged in vitro with p87-99. Administration of p87-99[91K > A], but not p87-99 [95T > A] or p87-99[96P > A], reduced the production of tumor necrosis factor (TNF)- alpha and interferon (IFN) gamma. IFN-gamma and TNF-alpha are two cytokines that are critical in the pathogenesis of EAE and MS.
Previous experiments from this laboratory have shown that Lewis rats were protected from experimental allergic encephalomyelitis (EAE) induced by the injection of myelin basic protein (MBP) in Freund's complete adjuvant if they were treated with the encephalitogenic peptide of MBP covalently linked to mouse anti-rat immunoglobulin (Ig) D. It was suggested that this protection developed because the antibody-peptide conjugate targeted the peptide to B cells and that this mode of presentation induced a Th2-like T cell response that controlled the concomitant encephalitogenic Th1 reaction to the autoantigen. The current experiments were carried out to test this hypothesis and to examine the alternative explanation for the protective effect of the conjugate pretreatment, namely that it induced a state of nonresponsiveness in the autoantigenspecific T cells. It was shown that EAE induction was suppressed in Lewis rats when the antibody-peptide conjugate was injected intravenously 14 and 7 d before immunization with MBP in adjuvant, but that anti-MBP antibody titers were at least as high in these animals as in controls that were not pretreated with the conjugate before immunization. Lymph node cells from these pretreated animals, while proliferating in vitro to MBP as vigorously as those from controls, produced less interferon gamma and were very inferior in their ability to transfer disease after this in vitro activation. In contrast, these same lymph node cells from protected rats generated markedly increased levels of messenger RNA for interleukin (IL)-4 and IL-13. When these in vitro experiments were repeated using the encephalitogenic peptide rather than MBP as the stimulus, the proliferative response of lymph node cells from pretreated donors was less than that from controls but was still readily detectable in the majority of experiments. Furthermore, the cytokine expression induced by the peptide was similar to that elicited by whole MBP. While these results support the original hypothesis that the anti-IgD-peptide conjugate pretreatment protected rats from EAE by inducing a Th2-type cytokine response, a totally unexpected finding was that this pretreatment greatly reduced the level of leukocyte infiltration into the central nervous system. This result provides a direct explanation for the protective effect of the pretreatment, but it raises questions regarding migratory and homing patterns of leukocytes activated by different immunological stimuli.
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Small changes in the peptide-major histocompatibility complex (MHC) molecule ligands recognized by antigen-specific T cell
receptors (TCRs) can convert fully activating complexes into partially activating or even inhibitory ones. This study examined
early TCR-dependent signals induced by such partial agonists or antagonists. In contrast to typical agonist ligands, both
an antagonist and several partial agonists stimulated a distinct pattern of zeta chain phosphorylation and failed to activate
associated ZAP-70 kinase. These results identify a specific step in the early tyrosine phosphorylation cascade that is altered
after TCR engagement with modified peptide-MHC molecule complexes. This finding may explain the different biological responses
to TCR occupancy by these variant ligands.
Transforming growth factors-beta (TGF-beta) modulate immune responses by inhibiting the proliferation of normal T lymphocytes. To examine the mechanism(s) of this inhibition, we studied the effect of TGF-beta 1 on selected events associated with the initiation and progression of the T lymphocyte cell cycle. Human peripheral blood T cells were stimulated with anti-CD3 mAb, PHA, PMA, or ionomycin, alone or in combination. TGF-beta 1 (0.5 to 10 ng/ml) partially inhibited the tyrosine phosphorylation of a 100-kDa protein, but not the calcium influx when cells were stimulated via TCR. Nuclear transcription of early activation genes (c-fos, c-jun, and c-myc) as determined by nuclear run-off assays, and steady state mRNA levels and/or protein products of intermediate activation genes (IL-2, IL-2R alpha, IL-2R beta, and transferrin receptor) were not affected by TGF-beta 1. Total cellular RNA synthesis and cell size after T cell stimulation were also not affected by TGF-beta 1. However, TGF-beta 1 inhibited the IL-2-dependent proliferation of Con A lymphoblasts by -50%. This inhibition was associated with the down-regulation of IL-2-mediated tyrosine phosphorylation of proteins of 120, 100, 85, 75, and 50 kDa. TGF-beta 1 also inhibited the IL-2-dependent phosphorylation of the retinoblastoma susceptibility gene product, which plays an important role in cell cycle progression. These results suggest that TGF-beta 1 inhibits T cell proliferation by down-regulating predominantly IL-2-mediated proliferative signals.
Experimental allergic encephalomyelitis (EAE) is an autoimmune disease characterized by inflammation and demyelination in the central nervous system. The effect of the immunosuppressive molecule transforming growth factor-beta, (TGF-beta 1) on chronic relapsing EAE produced by the transfer of myelin basic protein-specific T cell lines was studied. TGF-beta 1 markedly inhibited the activation and proliferation of myelin-basic protein-specific lymph node cells in vitro. This reduced the capacity of these cells to transfer EAE. In addition, administration of TGF-beta 1 in vivo consistently resulted in an improved clinical course, even when given during ongoing disease. Immunopathologic study demonstrated a marked reduction in central nervous system damage and expression of cell-surface lymphocyte function-associated Ag-1 and class II MHC molecules in TGF-beta 1-treated mice. These findings have identified TGF-beta 1 as a possible therapeutic agent for the human demyelinating disease multiple sclerosis.
The signals that determine the size and duration of the primary T cell immune response are not well defined. We studied CD4 T cells at an important checkpoint in their development: when they have become effectors and are ready to rapidly mediate effector functions, both via direct interaction with antigen (Ag)-presenting cells and via cytokine production. We determined the effects of specific Ag and the cytokines interleukin (IL) 2 and transforming growth factor (TGF) beta 1 on T helper cell type 2 (Th2) effector apoptosis versus expansion. Th2-polarized effector cells were generated in vitro from naive CD4 T of T cell receptor transgenic mice, and then restimulated with or without peptide Ag plus Ag-presenting cells and cytokines. In the absence of added cytokines, effector cells cultured without Ag died of apoptosis after 4-7 d. Paradoxically, Ag both induced proliferation and high levels of cytokine synthesis and accelerated effector cell death. IL-2 directly induced proliferation of effectors, supported and prolonged Ag-induced proliferation, and partially blocked apoptosis. TGF-beta did not effect proliferation or influence cytokine secretion, but it also partially blocked apoptosis. Together, IL-2 and TGF-beta synergized to almost completely block apoptosis, resulting in prolonged effector expansion and leading to the accumulation of a large pool of specific effectors. When Ag and both cytokines were present, the effector population increased 10(4)-10(5) fold over 20 d of culture. The synergy of IL-2 and TGF-beta suggests that they interfere with programmed cell death by distinct mechanisms. Since Th2 effectors are specialized to help B cells develop into antibody-secreting plasma cells, these results suggest that the availability of Ag and of the cytokines IL-2 and TGF-beta is a key factor influencing the fate of Th2 effector cells and thus the size and duration of the primary antibody response.
Experimental allergic encephalomyelitis (EAE) is an autoimmune disease characterized by inflammation and demyelination in the central nervous system (CNS). Administration of transforming growth factor-beta (TGF-beta) has been shown to inhibit EAE. In this study, the possible role of endogenous TGF-beta in the regulation of relapsing EAE produced by the transfer of myelin basic protein-specific T cell lines was assessed. Although TGF-beta is not present in the normal CNS, this cytokine was detected by immunohistology in areas of central nervous system inflammation in both acute and chronic disease. The administration of anti-TGF-beta at the disease onset led to a worsening of the clinical course of EAE and more extensive pathological lesions. These findings provide direct evidence for a role of endogenous TGF-beta in the remissions seen in chronic relapsing EAE.
The transforming growth factors (TGF) type β1 and β2 are regulatory cytokines strongly affecting rat astrocyte immune functions. Both cytokines suppressed presentation of autoantigen by astrocytes: highly encephalotogenic T cells cocultured with TGF-β-treated astrocytes in the presence of myelin basic protein did not become activated to transfer experimental allergic encephalomyelitis, a central nervous system (CNS) autoimmune disease. Furthermore, TGF-β1 and -β2 antagonized hyperinduction of astrocyte major histocompatibility complex (MHC) class II antigen expression by interferon-γ and tumor necrosis factor-α. Thus, TGF-β might be a potential regulator of CNS inflammation.
The feasibility of using T cell receptor (TcR) antagonist peptides to inhibit autoimmune disease has been examined. First, the fine antigenic structure of the I-As-restricted encephalitogenic determinant proteolipid protein (PLP) 139–151 has been analyzed. It was found that residues 145 and 148 were I-As anchor residues, and residue 144 appeared to be especially critical in T cell activation. Residues 142, 143, 146, and 147 were found to be crucial for activation of some, but not all, of the T cells studied. Next, good I-As-binding nonantigenic analogs were tested for TcR antagonism. Accordingly, several single substitution analogs were identified which could act as TcR antagonists. Moreover, when two such analogs were combined, the resulting TcR antagonist pool inhibited most of the PLP 139–151-specific T cell clones in vitro. When the efficacy of this TcR antagonist pool in inhibiting EAE induction in vivo was examined, it was found that the analog pool was a remarkably potent inhibitor of disease induction. The TcR antagonist pool was approximately 10-fold more potent than our best major histocompatibility complex blocker and was still capable of significant inhibition when injected in equimolar amounts with the encephalitogenic PLP 139–151 determinant.
Postcapillary endothelium at the sites of inflammation undergoes a series of changes collectively termed endothelial cell
activation. Activated endothelium expresses immunologically relevant surface proteins that include MHC class II antigens (Ags)
and adhesion proteins, as well as exhibits a number of functional changes. Endothelial activation has not been thoroughly
studied in CNS endothelium. We have examined cytokine-mediated endothelial activation in isolated rat CNS microvessels. Freshly
isolated rat CNS microvessels are viable in culture for at least 72 h. Untreated microvessels express no endothelial activation
antigens, but do exhibit constitutive expression of the transferrin receptor (tfR). INFγ induces a dose-dependent increase
in both MHC class II antigens and tfR measured by immunofluorescent staining and quantitated by laser cytometry. IFNγ-mediated
endothelial cell activation could be inhibited with as little as 2 ng/mL TGF-β1, although 100% inhibition was seen with 10
ng/mL TGF-β1. Cytokinepreactivated endothelial expression of class II Ag and tfR could also be inhibited by TGF-β1. TGF-β1-treated
microvessels become anergic to IFNγ stimulation. Results suggest that TGF-β1 may have a regulatory role in endothelial activation.
Previous studies have indicated that the cytokine transforming growth factor β1 (TGFβ1) has immunosuppressive properties and can inhibit the production of tumor necrosis factor (TNF) and Interleukin 1 (IL 1) by human peripheral blood mononuclear cells. In this study, we have examined the effects of TGFβ1 on the production of Interleukin 6 (IL 6) by human peripheral blood mononuclear cells. Treatment with only TGFβ1 leads to the induction of IL 6, and this was both dose- and time-dependent. The effect of TGFβ1 was evident at the level of IL 6 mRNA, suggesting TGFβ1-induced de novo synthesis of IL 6. Induction of IL 6 by TGFβ1 was specific, as other cytokines made by mononuclear cells (TNF and IL 1) were not induced by TGFβ1. Furthermore, when a panel of stimuli were compared for their ability to induce IL 1, TNF and IL 6 in the presence or absence of TGFβ1, IL 6 levels were augmented in the presence of TGFβ1, while the induction of IL 1 and TNF was inhibited significantly. These results indicate that TGFβ1 has complex effects on the production of cytokines by peripheral blood mononuclear cells and that TGFβ1 is not inhibitory for all cytokine production. The ability of TGFβ1 to induce IL 6 suggests that IL 6 may mediate some of the effects of TGFβ1.
To determine whether there is predominance of T cells expressing a particular TCR V beta chain in the inflammatory lesions of an autoimmune disease model, TCR expression was analyzed in central nervous system (CNS) tissues of mice with experimental allergic encephalomyelitis (EAE). Acute EAE was induced in SJL/J mice either by sensitization with a synthetic peptide corresponding to myelin proteolipid protein residues 139-151 or by adoptive transfer of myelin proteolipid protein peptide 139-151-specific encephalitogenic T cell clones. Mice were killed when they showed clinical signs of EAE or by 40 days after sensitization or T cell transfer. Cryostat CNS and lymphoid tissue sections were immunostained with a panel of mAb to T cell markers and proportions of stained cells were counted in inflammatory foci. In mice with both actively induced and adoptively transferred EAE, infiltrates consisted of many CD3+, TCR alpha beta+, and CD4+ cells, fewer CD8+ cells, and small numbers of TCR gamma delta+ cells. Approximately 30% of CD45+ leukocytes in the inflammatory foci were T cells. Cells expressing TCR V beta 2, 3, 4, 6, 7 and 14 were detected in the infiltrates, whereas TCR V beta 8 and 11, which that are deleted in SJL mice, were absent. When EAE was induced by transfer of T cell clones that use either V beta 2, 6, 10, or 17, there was also a heterogeneous accumulation of T cells in the lesions. Similar proportions of TCR V beta+ and gamma delta+ cells were detected in EAE lesions and in the spleens of the mice. Thus, at the time that clinical signs are present in acute EAE, peripherally derived, heterogeneous TCR V beta+ cells are found in CNS lesions, even when the immune response is initiated to a short peptide Ag or by a T cell clone using a single TCR V beta.
Experimental autoimmune encephalomyelitis (EAE) serves as an important animal model for understanding the events that lead to immune-mediated inflammation and tissue destruction within the central nervous system. We have utilized a murine adoptive transfer model of EAE and semiquantitative reverse transcriptase-polymerase chain reaction analysis to examine cytokine mRNA expression within the central nervous system in relation to the onset and resolution of paralysis associated with EAE. Spinal cord samples, obtained from mice as they progressed through discrete clinical stages of EAE, were examined for the expression of six cytokine genes (IL-1 alpha, IL-2, IL-4, IL-6, IL-10, and IFN-gamma). Distinct patterns of cytokine gene expression were observed during the acute, recovery, and chronic phases of EAE. The acute phase of disease was characterized by rapid increases in the levels of mRNA for IL-2, IL-4, IL-6, IFN-gamma, and IL-1 alpha. In fact, peak expression of several cytokine mRNA (e.g., IL-2, IL-4, IL-6, and IFN-gamma) occurred before the peak in clinical severity. In contrast, IL-1 alpha mRNA levels were elevated throughout the initial disease course. IL-10 mRNA demonstrated only modest increases during the acute phase of EAE. Stabilization of the clinical symptoms was characterized by rapid declines in the mRNA levels of IL-2, IL-4, IL-6, and IFN-gamma. The decreases in these four cytokine mRNA levels occurred concomitant with a dramatic rise in IL-10 mRNA. Finally, of the six cytokine mRNA examined, only IL-1 alpha, IFN-gamma, and IL-10 mRNA remained elevated during the early chronic stage. These results suggest that local cytokine production varies significantly during the course of EAE and that increases in discrete sets of cytokines are associated with the acute response and the recovery/chronic phase of disease.
Nylon wool adherent, CD4+ T cells from the spleens of rats that have recovered from experimental autoimmune encephalomyelitis (EAE) inhibit the in vitro production of IFN-gamma, but not IL-2, by effector cells of EAE when cocultured in the presence of myelin basic protein Ag. When anti-transforming growth factor-beta (TGF-beta) antibodies are added to the co-cultures, IFN-gamma production is restored to normal levels. Irrelevant control antibodies have no effect. The same pattern of response was obtained with cells incubated in serum-free medium. In other experiments, purified TGF-beta was added to cultures of effector cells in the presence of antigen. TGF-beta inhibited the production of IFN-gamma by these cells in a dose-dependent manner, but had no apparent inhibitory effect on IL-2 production. Finally, supernatants from cultures containing effector cells and CD4+ suppressor cells plus Ag contained measurable amounts of TGF-beta, whereas supernatants from cultures of effector cells plus Ag contained no measurable amounts of TGF-beta. These results suggest that CD4+ Ts cells of EAE regulate effector cells of EAE through a mechanism that involves the secretion of TGF-beta and that the inhibitory function of this cytokine can be reversed with neutralizing antibodies directed against TGF-beta.
A strategy for the isolation of mononuclear cells from the brain parenchyma of mice with ongoing central nervous system (CNS) inflammation has been developed in order to permit flow cytometric (FCM) analysis of these cell populations. Sindbis virus (SV) encephalitis in mice is characterized morphologically by an infiltration of mononuclear cells into both brain parenchyma and cerebrospinal fluid (CSF). Perfused brain tissue from infected animals is collected, homogenized, and subjected to a mild enzymatic digestion. A sedimentation at unit gravity is performed to remove any large particulate debris, and the remaining tissue is then centrifuged over a modified density gradient which separates intact cells from smaller tissue fragments. Cells collected directly from these gradients can be stained with monoclonal antibodies and analyzed by FCM without further manipulation. Data generated by this method correlates with previous studies of SV encephalitis using immunohistochemical analysis of brain tissue sections to quantify mononuclear cell types. This suggests that representative samples of the cellular infiltrate are obtained using this technique. The approach however, offers the possibility of more sophisticated and quantitative analyses of CNS inflammatory cells which is unobtainable by tissue section staining.
Myelin basic protein (MBP) or helper T cells reactive against MBP induce an autoimmune disease, experimental allergic encephalomyelitis, in B10.PL and PL/J inbred mice. In both strains, virtually the entire repertoire of MBP-specific T cells recognize an N-terminal peptide fragment in the context of the I-Au molecule encoded by the major histocompatibility complex (MHC) and utilize a very limited set of T-cell receptor genes. To delineate the nature of the trimolecular complex, consisting of the T-cell receptor, MBP-peptide fragment, and MHC molecule (I-Au), we have synthesized 13 variants of the 9-mer N-terminal immunodominant peptide differing at residue 4 and studied their immune recognition in vitro and in vivo. These substitutions have a striking range of effects on T-cell activation, ability to bind to the MHC molecule, and initiation of immune responses in vivo. An understanding of the autoimmune peptide/MHC/T-cell receptor interactions allowed us to design variant 9-mer peptides that have high affinity for an MHC molecule and are effective in blocking experimental allergic encephalomyelitis, possibly through two distinct mechanisms, peripheral T-cell tolerance and the inhibition of binding of the encephalitogenic peptide to the MHC molecules.
Recombinant human transforming growth factor-beta (TGF-beta) was found to inhibit the adhesive phenotype of human umbilical vein endothelial cells for human PBL, purified T lymphocytes, and PHA-activated lymphoblasts. TGF-beta inhibited lymphocyte attachment to resting human umbilical vein endothelial cells and also to endothelial monolayers stimulated with the pro-inflammatory cytokines TNF-alpha and IL-1 beta. Our investigations also show that the ability of endothelial cells to respond to TGF-beta by altering their adhesiveness is lost with prolonged culture of the cells. However, this loss is selective as TGF-beta inhibits cell proliferation in both early and late passage endothelial cells. These results suggest that in vivo TGF-beta may inhibit the adhesive phenotype of endothelial cells and also may limit the immunologic response occurring at the endothelial cell barrier.
Experimental allergic encephalomyelitis (EAE) is an autoimmune disease characterized by inflammation and demyelination in the central nervous system. The effect of the immunosuppressive molecule transforming growth factor-beta, (TGF-beta 1) on chronic relapsing EAE produced by the transfer of myelin basic protein-specific T cell lines was studied. TGF-beta 1 markedly inhibited the activation and proliferation of myelin-basic protein-specific lymph node cells in vitro. This reduced the capacity of these cells to transfer EAE. In addition, administration of TGF-beta 1 in vivo consistently resulted in an improved clinical course, even when given during ongoing disease. Immunopathologic study demonstrated a marked reduction in central nervous system damage and expression of cell-surface lymphocyte function-associated Ag-1 and class II MHC molecules in TGF-beta 1-treated mice. These findings have identified TGF-beta 1 as a possible therapeutic agent for the human demyelinating disease multiple sclerosis.
Transforming growth factor-beta (TGF-beta) has been shown to regulate the proliferation and function of several different cell types in the immune system. We have examined the effect of TGF-beta on the proliferation of murine macrophages in liquid culture. TGF-beta by itself did not induce proliferation of differentiated (7 days in culture) bone marrow-derived macrophages (BMM). In the presence of M-CSF, TGF-beta enhanced the proliferation of differentiated BMM and elicited peritoneal macrophages but had an inhibitory effect on the proliferation of nonadherent BMM (3 days in culture). The effect of TGF-beta was not restricted to M-CSF-dependent proliferation but was also observed for GM-CSF-dependent proliferation. The autocrine production of TGF-beta appeared to contribute to the proliferation of BMM. The addition of antibody against TGF-beta inhibited M-CSF- and GM-CSF-dependent proliferation 32% and 28%, respectively. In bone marrow, TGF-beta may be an important negative regulator of macrophage proliferation; whereas, in the tissues, TGF-beta may enhance macrophage proliferation.
Amino acid sequence homology was found between viral and host encephalitogenic protein. Immune responses were then generated in rabbits by using the viral peptide that cross-reacts with the self protein. Mononuclear cell infiltration was observed in the central nervous systems of animals immunized with the viral peptide. Myelin basic protein (MBP) is a host protein whose encephalitogenic site of ten amino acids induces experimental allergic encephalomyelitis. By computer analysis, hepatitis B virus polymerase (HBVP) was found to share six consecutive amino acids with the encephalitogenic site of rabbit MBP. Rabbits given injections of a selected eight- or ten-amino acid peptide from HBVP made antibody that reacted with the predetermined sequences of HBVP and also with native MBP. Peripheral blood mononuclear cells from the immunized rabbits proliferated when incubated with either MBP or HBVP. Central nervous system tissue taken from these rabbits had a histologic picture reminiscent of experimental allergic encephalomyelitis. Thus, viral infection may trigger the production of antibodies and mononuclear cells that cross-react with self proteins by a mechanism termed molecular mimicry. Tissue injury from the resultant autoallergic event can take place in the absence of the infectious virus that initiated the immune response.
PLP is the major protein constituent of central nervous system myelin. We have previously shown that SJL/J (H-2s) mice develop an acute form of EAE after immunization with PLP. The purpose of the present study was to identify an encephalitogenic determinant of PLP for SJL mice. We immunized SJL/J mice with a synthetic peptide identical to residues 130-147 QAHSLERVCHCLGKWLGH of murine PLP, a sequence having an amphipathic alpha-helical conformation. Although it did not induce disease, an overlapping peptide containing residues 139-154 HCLGKWLGHPDKFVGI was encephalitogenic. Immunization with this peptide induced severe clinical and histologic EAE in 3 of 20 mice. T cell enriched ILN cells from these mice responded specifically (3H-thymidine incorporation) to this peptide as well as to shorter analogues of this domain containing serine in place of cysteine at residues 138 and 140. Immunization with the serine-substituted PLP peptides 137-151 VSHSLGKWLGHPDKF and 139-151 HSLGKWLGHPDKF induced severe, acute EAE in 4 of 9 and 15 of 15 SJL mice, respectively, and their T cell enriched ILN cells responded not only to the analogues, but also to the native PLP sequence 139-154. These results indicate that residues 139-151 of murine PLP is an encephalitogenic determinant for SJL mice. Furthermore, like the PLP encephalitogenic domain for SWR (H-2q) mice, this determinant is also a T cell epitope with a coding sequence at the end of an exon.
The molecular mimicry represented by cross-recognition of determinants shared by unrelated antigens by antibodies or T cells is of broad immunological interest. In this study, we analyzed the cross-recognition by CD4+ T cells of a peptide epitope shared by two mycobacterial proteins of diverse sequence, represented by the 19-kDa antigen of Mycobacterium tuberculosis and the 28-kDa antigen of Mycobacterium leprae. This epitope was immunodominant with respect to the 19-kDa antigen, but cryptic in relation to the 28-kDa antigen. The cross-reactive epitope cores were identified by Pepscan window analysis and found to be eight residues long in both antigens (residues 69-76 and 127-134). Alignment of these octameric sequences revealed two identical and five conservatively related amino acids. Within the epitope core, two residues (73Asn and 76Ile) were identified as critical for recognition on the basis of inhibition of the cross-reactive T cell proliferative response using singly substituted analog peptides. These results suggest that T cell cross-reactive epitopes can exist in proteins with apparently not more than random levels of sequence homology. Their potential for unsuspected cross-sensitization may play a role in the maintenance of T cell memory, in the pathogenesis of autoimmune diseases and possibly in a wide range of host immune responses to infectious pathogens.
A study was made of the ability of the superantigen staphylococcal enterotoxin B (SEB) to induce relapses of experimental allergic encephalomyelitis (EAE) in SJL mice that had partially or completely recovered from acute EAE. We find that a single injection of 0.05 mg SEB i.v. induces mild relapses in 50% of such mice. In addition, tumor necrosis factor (TNF)-alpha (0.2 micrograms, i.p.) also induces EAE relapses in 43% of SJL mice when injected 1-2 months after recovery. SEB does not induce a second relapse if reinjected when V beta 17a+T cells are still partially deleted. In these mice, however, TNF-alpha is equally effective in inducing relapses as in mice that did not receive SEB previously. We showed earlier that transforming growth factor (TGF)-beta and TNF-alpha have antagonistic effects on experimental autoimmune diseases; e.g., in spontaneously relapsing EAE, TGF-beta and anti-TNF were protective, while anti-TGF-beta caused disease exacerbation. Interleukin (IL)-10 is also known to counteract certain TNF effects. We now find that both human IL-10 and TGF-beta 2 lower the incidence of EAE relapses when given simultaneously with SEB or TNF-alpha. The protective effect of TGF-beta is significant only against relapses induced by SEB (reduced to 9%), and that of IL-10 only against relapses induced by TNF (reduced to 0%) with the treatment regimens employed. Neutralizing anti-TGF-beta does not increase the incidence of SEB-induced EAE relapses. In contrast, anti-IL-10 increases both the incidence and the severity of such relapses. We conclude that TNF production is probably important in causing EAE relapses, but that other aspects of the SEB-induced reactivation of myelin-specific T cells also contribute. Furthermore, endogenous IL-10 rather than TGF-beta production appears to limit the susceptibility to induction of EAE relapses in this model.
We have been studying the suppression of experimental autoimmune encephalomyelitis in the Lewis rat after oral administration of myelin basic protein (MBP). Suppression is mediated by CD8+ T cells that adoptively transfer protection and suppress immune responses in vitro. This suppression is mediated by secretion of TGF-beta following triggering by the fed antigen. In the present study, we tested the ability of overlapping 20 amino acid peptides from MBP to trigger suppression mediated by spleen cells from Lewis rats orally tolerized to MBP. Using a transwell system, we found that spleen cells from MBP orally tolerized animals stimulated by residues 21-40, 51-70 and 101-120 of MBP suppress proliferative responses of an ovalbumin specific cell line. This suppression correlated with secretion of TGF-beta by cells stimulated with the peptide. In addition, T cells from animals fed the tolerogenic peptide 21-40 alone secreted TGF-beta whereas no TGF-beta release or in vitro suppression was observed in animals fed the MBP encephalitogenic determinant 71-90. The 71-90 peptide triggered proliferation of MBP primed cells from animals immunized with MBP/CFA whereas the suppressor epitopes identified above did not. Furthermore, oral administration of peptide 21-40 suppressed disease induced by peptide 71-90. DTH responses to 71-90 were not affected by oral administration of peptide 21-40 whereas DTH responses to whole MBP were suppressed. These results demonstrate that distinct suppressor determinants exist on MBP which are separate from encephalitogenic determinants, and that epitope-driven bystander suppression plays an important role in down-regulation of tissue specific autoimmune processes following oral tolerization. These findings have important implications for the design of tissue specific targeted immunotherapy by oral tolerization in humans.
We previously reported that the CD4+ suppressor cells (Ts) that regulate recovery of Lewis rats from experimental autoimmune encephalomyelitis (EAE) produce transforming growth factor-beta (TGF-beta). We also reported that TGF-beta downregulates interferon-gamma (IFN-gamma), but not interleukin-2 (IL-2) production, by the CD4+ effector T cells (Te) that mediate EAE. We now report that TGF-beta 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-beta inhibited TNF/LT production in a dose-dependent fashion. Moreover, neutralizing anti-TGF-beta antibodies augmented TNF/LT production in the Te+MBP cultures. We also confirm that TGF-beta inhibits adoptive transfer of EAE. In contrast, murine IL-10 only partially inhibited TNF/LT and IFN-gamma production by Te. We conclude that TGF-beta production by Ts plays a major role in recovery from EAE in the Lewis rat by inhibiting TNF/LT and IFN-gamma production by the effector cells that mediate EAE.
Experimental allergic encephalomyelitis (EAE) is an autoimmune disease characterized by central nervous system inflammation and demyelination. Retinoids regulate cell differentiation and growth by binding to and activating retinoic acid receptors, which seem to be nuclear transcription factors. The effect of retinoids on chronic relapsing EAE produced by the transfer of myelin basic protein (MBP)-specific lymph node cells (LNC) was studied. All-trans-retinoic acid (tRA) inhibited the proliferation of MBP-specific LNC in vitro. However, the capacity of these cells to transfer EAE was markedly reduced by concentrations of tRA that only mildly inhibited T cell proliferation. The presence of tRA during in vitro MBP-specific LNC activation resulted in a considerable increase in IL-4 mRNA, whereas mRNA for IL-2, TNF-alpha, and IFN-gamma was decreased. Increased IL-4 also was detected in culture supernatants. However, the presence of a neutralizing Ab to IL-4 (11B11) during MBP-specific LNC activation in vitro did not reverse the inhibition of encephalitogenicity caused by tRA. The administration of retinoids in vivo resulted in an improved clinical course, even when given after disease onset. These findings suggest that T cell activation in the presence of tRA results in the development of T cells of the Th2 phenotype, which, in turn, might be responsible for the decrease in the encephalitogenicity of MBP-specific T cells. The modulation by retinoids of an immune response dominated by Th1-like T cells to one in which the protective cytokines of Th2-like cells predominate may have potential relevance for human demyelinating diseases such as multiple sclerosis.
Structural similarity between viral T cell epitopes and self-peptides could lead to the induction of an autoaggressive T cell response. Based on the structural requirements for both MHC class II binding and TCR recognition of an immunodominant myelin basic protein (MBP) peptide, criteria for a data base search were developed in which the degeneracy of amino acid side chains required for MHC class II binding and the conservation of those required for T cell activation were considered. A panel of 129 peptides that matched the molecular mimicry motif was tested on seven MBP-specific T cell clones from multiple sclerosis patients. Seven viral and one bacterial peptide efficiently activated three of these clones. Only one peptide could have been identified as a molecular mimic by sequence alignment. The observation that a single T cell receptor can recognize quite distinct but structurally related peptides from multiple pathogens has important implications for understanding the pathogenesis of autoimmunity.
We demonstrate that cognate peptide ligands altered at T cell receptor (TCR) contact residues and bound to class II major histocompatability complex can change the cytokine pattern of mature T cell clones. Myelin basic protein peptide 85-99-reactive Th0 T cell clones were stimulated with altered peptide ligands, which acted both as TCR antagonist and induced new mRNA synthesis and protein secretion of TGF-beta 1, while no longer inducing mRNA synthesis or protein secretion of IL-2, IL-4, IL-10, and IFN gamma. The modified peptides failed to induce a detectable calcium flux, p56lck activation, or thymidine incorporation, yet triggered nearly equal amounts of IL-4 secretion in the presence of ionomycin. Antigen-induced modulation of T cell cytokine secretion may be important in regulating the immune response.
The T cell receptor (TCR) can interact with a spectrum of peptides as part of its ligand, including the immunogenic peptide, variants of this peptide,and apparently unrelated peptides. The basis of this broad specificity for ligand was investigated by substitution analysis of a peptide antigen and functional testing using a B cell apoptosis assay. A peptide containing as few as 1 aa in common with this peptide could stimulate a specific T cell response. Two endogenous ligands, an agonist and a partial agonist, were readily identified from a search of the SwissProt database, indicating that multiple endogenous ligands likely exist for a given T cell. These findings strongly support the concept that one TCR has the ability to interact productively with multiple different ligands, and provide evidence that such ligands exist in the endogenous peptide repertoire.
By immunizing Lewis rats with myelin basic protein (MBP) peptide variants derived from the major encephalitogenic epitope of guinea pig (MBP(68-88) and then isolating encephalitogenic T cells from these animals, we demonstrated that the variant peptides do not elicit the same encephalitogenic T cell subsets as those induced by the wild-type peptide or by intact MBP. Rather, the pathogenic T cells differed in clonal composition as reflected by their heterogeneous responses to a panel of variant peptides and by their T cell receptor usage. Thus, molecules mimicking the MBP(68-88) autoantigen can elicit pathogenic T cell subsets without necessarily cross-reacting with T cells specific for the original autoantigen. This suggests that a more clonally diverse group of pathogenic T cells might be involved in EAE than has been apparent from studies with intact MBP or its unaltered peptides.
Experimental autoimmune encephalomyelitis (EAE) is an autoimmune disease induced by immunization with myelin basic protein (MBP), proteolipid protein, or encephalitogenic peptides from these myelin components. EAE resembles basic protein multiple sclerosis in some of its clinical and histologic features, and serves as an experimental model for this and other autoimmune diseases. In this study, we examine i.v. peptide therapy of EAE in detail, and show that repeated i.v. injections of MBP peptides effectively treat EAE in (PLJxSJL)F1 mice. In this study, administration of the immunodominant epitope (MBP Ac1-11) prevents MBP-induced disease, whereas the subdominant epitope MBP 31-47 is neither required nor sufficient. Intravenous administration of substituted MBP peptide analogues is also effective in treating EAE, provided the peptide side chains presumed to be involved in TCR contact and MHC binding are preserved. A substituted MBP peptide analogue that forms long-lived peptide-MHC complexes in vivo is more effective than the unmodified MBP peptide. Lower doses of the substituted peptide analogue are effective, and the effect is longer lasting than treatment with the unmodified peptide. Clinical signs of EAE are reversed by injection of the substituted peptide during the acute phase of disease. Moreover, treatment of mice in the remission phase of EAE results in a dramatically reduced incidence of relapse. In summary, we have shown that EAE can be reversed after onset and treated during remission with an MBP peptide analogue that has been modified for improved therapeutic potency.
Differential activation of CD4+ T-cell precursors in vivo leads to the development of effectors with unique patterns of lymphokine secretion. To investigate whether the differential pattern of lymphokine secretion is influenced by factors associated with either the display and/or recognition of the ligand, we have used a set of ligands with various class II binding affinities but unchanged T-cell specificity. The ligand that exhibited approximately 10,000-fold higher binding to I-Au considerably increased the frequency of interferon gamma-producing but not interleukin (IL) 4- or IL-5-secreting cells in vivo. Using an established ligand-specific, CD4+ T-cell clone secreting only IL-4, we also demonstrated that stimulation with the highest affinity ligand resulted in interferon gamma production in vitro. In contrast, ligands that demonstrated relatively lower class II binding induced only IL-4 secretion. These data suggest that the major histocompatibility complex binding affinity of antigenic determinants, leading to differential interactions at the T cell-antigen-presenting cell interface, can be crucial for the differential development of cytokine patterns in T cells.
In experimental autoimmune encephalomyelitis (EAE) induced with myelin proteolipid protein (PLP) peptide 139-151, we have previously shown that the disease is mediated by Th1 cells, which recognize tryptophan 144 as the primary TCR contact point. Here we describe an altered peptide ligand (APL), generated by a single amino acid substitution (tryptophan to glutamine) at position 144 (Q144), which inhibits the development of EAE induced with the native PLP 139-151 peptide (W144). We show that the APL induces T cells that are cross-reactive with the native peptide and that these cells produce Th2 (IL-4 and IL-10) and Th0 (IFN gamma and IL-10) cytokines. Adoptive transfer of T cell lines generated with the APL confer protection from EAE. These data show that changing a single amino acid in an antigenic peptide can significantly influence T cell differentiation and suggest that immune deviation may be one of the mechanisms by which APLs can inhibit an autoimmune disease.
In the present study, we demonstrate that TGF-beta is capable of enhancing macrophage ability to produce IL-10 in normal and EL4 tumor-bearing mice. We found the increase in IL-10 in ascitic fluid and IL-10 mRNA expression in macrophages in parallel with the TGF-beta level and tumor progression. The macrophage production of IL-10 in the tumor-bearing mice was significantly enhanced without LPS stimulation in vitro, compared with normal controls. To clarify the mechanism wherein increased IL-10 production was induced, anti-TGF-beta or anti-IL-10 Abs were administered to EL4-bearing mice. Administration of anti-TGF-beta Ab led to a reduction in the IL-10 contents in ascitic fluid of tumor-bearing mice; however, anti-IL-10 Ab administration did not prevent the increase in TGF-beta contents. Enhanced IL-10 production and mRNA expression of macrophages from the tumor-bearing mice were also reduced by anti-TGF-beta Ab administration. Both anti-TGF-beta and anti-IL-10 Ab administration restored the TNF-alpha production by macrophages in EL4-bearing mice. In normal macrophages, in vitro pretreatment with TGF-beta 1 potentiated IL-10 production, and when natural TGF-beta 1 was administered to normal mice, the recovered peritoneal macrophages showed enhanced IL-10 production. Based on the above findings it can be concluded that TGF-beta enhances macrophage ability to produce IL-10, which sheds a new light on the role of TGF-beta in the immune system.
Migration of leukocytes through an in vitro, cell culture model of the blood-brain barrier (BBB) composed of murine brain microvessel endothelial (En) cells and astrocytes, and in vivo in experimental allergic encephalomyelitis (EAE), was investigated. We have recently shown that the adhesiveness of cultured murine brain microvascular endothelial cells for lymphocytes can be increased significantly by pretreatment with IL-1 beta, TNF-alpha, IFN-gamma, and LPS. In the present study, we investigated the role of TGF-beta 2 on the migration of leukocytes through the BBB. In vitro migration was assessed by measuring the percentage of 51Cr-labeled leukocytes migrating through the En/astrocyte monolayers. The basal level of migration was up-regulated significantly by treating the En/astrocyte monolayers with IL-1 alpha, IFN-gamma, TNF-alpha, and LPS. The ability of these cytokines to modulate migration was dose-dependent. Treatment of En cell/astrocyte monolayers with TGF-beta 2 down-regulated the level of leukocyte migration up-regulated by IL-1 alpha, IFN-gamma, and TNF-alpha in vitro in a dose-dependent manner. TGF-beta 2 also inhibited the migration of lymphocytes into the central nervous system (CNS) in vivo in a dose-dependent fashion. Taken together, these findings strongly suggest that TGF-beta plays an important role in the reduction of lymphocyte infiltration into the CNS in inflammatory demyelinating diseases such as EAE.
It had been demonstrated previously that the administration of transforming growth factor-beta 1 (TGF-beta 1) reduced the clinical severity of experimental allergic encephalomyelitis (EAE). Treatment with the related immunosuppressive molecule, TGF-beta 2, resulted in similar inhibition of T cell activation and proliferation in vitro. Long-term treatment was effective in reducing clinical severity of EAE and the number of relapses in mice receiving either myelin basic protein- or peptide-91-103-specific T cell lines. When examined histologically, mice that had received TGF-beta 2 demonstrated significantly less inflammation and demyelination in the central nervous system. Examination of other organs demonstrated no pathology or deleterious side effects from long-term TGF-beta 2 therapy. These findings have relevance for the use of TGF-beta 2 as a therapeutic agent for the human demyelinating disease, multiple sclerosis.
Astrocytes participate in the pathophysiology of central nervous system (CNS) inflammatory disease. Astrocyte expression of adhesion molecules, cytokines, and major histocompatibility complex antigens may contribute to these inflammatory processes. In addition, recent data suggested that astrocytes may be a source of monocyte chemoattractant protein-1 (MCP-1). MCP-1 is a member of the chemokine family of small cytokines and functions both as a chemoattractant as well as a stimulator of monocytes. To further characterize the role of astrocytes in CNS inflammation, we examined the effect of inflammatory cytokines on MCP-1 expression by astrocytes. Results of these studies demonstrate that the pro-inflammatory cytokine tumor necrosis factor alpha (TNF alpha) upregulates MCP-1 message and protein expression. The pleiotropic cytokine transforming growth factor beta (TGF beta) also stimulated MCP-1 expression. When astrocytes were exposed to both cytokines simultaneously, an additive effect on MCP-1 message, but not MCP-1 protein expression, was observed. These data suggest that TNF alpha and TGF beta, each present during CNS inflammatory disease, may upregulate the expression of MCP-1 which, in turn, may function to both recruit monocytes to the site of inflammation as well as to activate those monocytes already present in an inflammatory lesion.
Resistance to experimental allergic encephalomyelitis (EAE) induction by homogenized myelin (MSCH) in complete Freund's adjuvant (CFA) and pertussigen (P) in SJL mice was seen 1 week after intravenous injection of PLP 139-151 coupled to spleen cells (PLP-ECDI-SP). Although this resistance could be transferred by spleen cells enriched for CD8+ T cells and thus had a component of immunoregulatory T cells, it was primarily due to anergy, as it was reversible by four daily injections of interleukin (II)-2 starting 3 days after the PLP-ECDI-SP. Earlier treatment with IL-2 did not reverse the tolerance. In view of the known higher sensitivity to anergy induction of Th1 than of Th2 cells, a change in the cytokine balance in the response to MSCH+CFA after anergy induction might be responsible for the resistance to EAE induction. The effect of treatment with cytokines alone on induction of EAE was therefore also determined. Short-term (1-2 weeks) daily pretreatment with IL-2 (4000 U) or TGF-beta 2 (1 micrograms) somewhat decreased the susceptibility to subsequent EAE induction, but IL-4 (5 ng), IL-10 (5 micrograms) or IL-12 (50-200 ng) had no effect under those conditions, even if low doses of PLP were injected simultaneously. Daily injections of IL-4 over an 8-week period prior to immunization, however, significantly lowered the incidence of EAE. Simultaneous injections of IFN-gamma (2000 U/day) completely abolished this effect of IL-4. The effect of these cytokines administered immediately after the immunization with MSCH + CFA + P was also examined. As shown earlier, TGF-beta 2 (100-1000 ng/day) caused a marked protection when it was given intraperitoneally on days 5-9 after injection of MSCH + CFA. IL-4 (5 ng/day), in contrast, was very protective when administered on days 0-4 and less so when given on days 5-9 or even on days 0-12. IL-10 (1 microgram/day) was not protective under these conditions and IL-12 (50 ng/day) significantly increased the severity and mortality of EAE when given on days 0-4 after MSCH + CFA.
Investigations of T-cell responses to altered peptide ligands have provided functional evidence that a T-cell receptor can interpret subtle structural changes in its ligand, highlighting the complexity of this antigen receptor signaling system. Over the past year, observations from many studies have suggested several roles for such analog peptides in various aspects of immune responses. Collectively, these data strongly suggest the existence of naturally occurring altered peptide ligands in the endogenous peptide repertoire, that can actively participate in the development and shaping of T-cell immunity.
Postcapillary endothelium at the sites of inflammation undergoes a series of changes collectively termed endothelial cell activation. Activated endothelium expresses immunologically relevant surface proteins that include MHC class II antigens (Ags) and adhesion proteins, as well as exhibits a number of functional changes. Endothelial activation has not been thoroughly studied in CNS endothelium. We have examined cytokine-mediated endothelial activation in isolated rat CNS microvessels. Freshly isolated rat CNS microvessels are viable in culture for at least 72 h. Untreated microvessels express no endothelial activation antigens, but do exhibit constitutive expression of the transferrin receptor (tfR). INF gamma induces a dose-dependent increase in both MHC class II antigens and tfR measured by immunofluorescent staining and quantitated by laser cytometry. IFN gamma-mediated endothelial cell activation could be inhibited with as little as 2 ng/mL TGF-beta 1. although 100% inhibition was seen with 10 ng/mL TGF-beta 1. Cytokine-preactivated endothelial expression of class II Ag and tfR could also be inhibited by TGF-beta 1. TGF-beta 1-treated microvessels become anergic to IFN gamma stimulation. Results suggest that TGF-beta 1 may have a regulatory role in endothelial activation.
The inflammatory nature of multiple sclerosis (MS) implicates the participation of immunoregulatory cytokines, including the T-helper type 1 (Th1) cell-associated interferon-gamma (IFN-gamma), the Th2 cell-related interleukin-4 (IL-4), and the immune response-downregulating cytokine transforming growth factor-beta (TGF-beta), but proof for their involvement in MS has been lacking. By adopting in situ hybridization with complementary DNA oligonucleotide probes for human IFN-gamma IL-4, and TGF-beta, the expression of mRNA for these cytokines was detected in mononuclear cells (MNC) from blood and cerebrospinal fluids. Strongly elevated levels of MNC expressing all three cytokines were found in peripheral blood and at even higher frequencies in cerebrospinal fluid from untreated patients with MS and optic neuritis, i.e., a common first manifestation of MS, compared with patients with other neurological diseases and healthy subjects. In MS and optic neuritis, IL-4 mRNA expressing cells predominated, followed by TGF-beta- and IFN-gamma-positive cells. Control patients with myasthenia gravis had similarly elevated levels of IFN-gamma and TGF-beta and TGF-beta mRNA expressing blood MNC but lower numbers of IL-4-positive cells. No or slight disability of MS was associated with high levels of TGF-beta mRNA expressing cells, while MS patients with moderate or severe disability had high levels of IFN-gamma-positive cells. IFN-gamma and TGF-beta may have opposing effects in MS, and treatments inhibiting IFN-gamma and/or promoting TGF-beta might ameliorate MS.