Ahmad Al-Sabbagh

Suppression of experimental autoimmune encephalomyelitis by oral administration of myelin basic protein: III. Synergistic effect of lipopolysaccharide☆

Oral administration of myelin basic protein (MBP) suppresses experimental autoimmune encephalomyelitis (EAE) in Lewis rats immunized with MBP in Freunds adjuvant. The immunomodulator bacterial lipopolysaccharide (LPS) when given orally in conjunction with MBP enhances the protective effects of MBP feeding in EAE. This synergy was achieved only following oral administration of LPS but not following subcutaneous injection. In contrast, subcutaneous administration of LPS abrogated oral tolerance. A synergism between oral LPS and MBP was also demonstrated for antigen-specific suppression of delayed type hypersensitivity (DTH) responses. Antibody responses to MBP were suppressed by oral administration of MBP but not by MBP plus LPS. The lipid A moeity of LPS mimicked the effects of LPS on disease protection and DTH suppression. These data demonstrate that adjuvants can enhance the induction of antigen-specific oral tolerance for suppression of cell-mediated experimental autoimmune responses.

Suppression of experimental autoimmune encephalomyelitis by oral administration of myelin basic protein. V. Hierarchy of suppression by myelin basic protein from different species

We have been investigating the suppression of experimental autoimmune encephalomyelitis (EAE) by oral tolerization to autoantigens. In the present study the tolerizing effect of orally administered myelin basic protein (MBP) from different species was examined in the Lewis rat, Hartley guinea pig, and SJL/J mouse model of EAE. Animals were fed guinea pig, rat, bovine, human or mouse-MBP and then immunized with the homologous species of MBP or myelin: Lewis rats were immunized with rat MBP, Hartley guinea pigs with guinea pig-MBP, and SJL/J mice with mouse myelin. Clinical expression of EAE and delayed-type hypersensitivity (DTH) responses to MBP were assessed. In each species, suppression of disease and DTH responses were most pronounced by tolerization with the homologous species of MBP. In addition, cross-species tolerization was observed in each species and in general was less suppressive than homologous MBP although in some instances MBP from a heterologous species was as effective as tolerization with the homologous species. We also studied guinea pig-MBP induced EAE in the Lewis rat because it is a widely studied model of EAE and found that oral tolerization with guinea pig MBP was as suppressive as rat MBP. Of note is that oral tolerization with mouse MBP suppressed myelin-induced EAE in the SJL mouse in which autoimmunity to proteolipid protein appears to play a primary role, suggesting that antigen-driven bystander suppression following oral tolerization with autoantigens (Miller et al., 1991b) may be an important contributing mechanism for suppression of EAE following oral tolerization with MBP in this model.(ABSTRACT TRUNCATED AT 250 WORDS)

Oral Tolerance: A Biologically Relevant Pathway to Generate Peripheral Tolerance against External and Self Antigens

OT is a relevant biological pathway for generating peripheral tolerance against both self and external antigens with minimal side effects (fig. 3). This route might, therefore, contain promising potential for the treatment of autoimmune and allergic diseases in the human (fig. 3). Thus, oral administration of autoantigens suppresses experimental autoimmune diseases (EAE, EAU, AA, collagen-induced arthritis, NOD diabetes) in a disease- and antigen-specific manner, and oral administration of alloantigens has led to increase of allograft survival. OT might be important in treatment of immune complex diseases and food allergies. OT is mediated by T lymphocytes using at least two nonmutually exclusive mechanisms: suppression and anergy. Suppression can be adoptively transferred by CD8+ T lymphocytes which act by releasing TGF-beta and IL-4 following antigen-specific triggering. Antigen-driven tissue-directed suppression occurs following oral administration of an antigen from the target organ, even if it is not the disease-inducing antigen (bystander suppression). Thus, synthetic peptides can induce OT, and tolerogenic epitopes of antigen may be different from the autoreactive epitope. Due to the promising results in animal models, OT is being tested in clinical trials in multiple sclerosis, rheumatoid arthritis and uveitis [193, 194].

Suppression of experimental autoimmune encephalomyelitis by oral administration of myelin basic protein. VI. Suppression of adoptively transferred disease and differential effects of oral vs. intravenous tolerization.

Antigen-driven tolerance is an effective method of suppressing cell-mediated immune responses. We have previously shown that oral administration of myelin basic protein (MBP) suppresses experimental autoimmune encephalomyelitis (EAE) when it is actively induced by MBP emulsified in complete Freunds adjuvant. In order to further study antigen-driven tolerance in this model, we investigated the effect of oral tolerization on adoptively transferred EAE and compared oral tolerance to intravenously (i.v.) administered MBP in both actively induced EAE and adoptively transferred EAE. Although orally tolerized animals were not protected from adoptively transferred EAE, spleen cells from orally tolerized animals suppressed adoptively transferred EAE when co-transferred with encephalitogenic cells or when injected into recipient animals at a different site at the time encephalitogenic cells were transferred. This suppression was mediated by CD8+ T cells, correlated with suppression of DTH responses to MBP, and was associated with decreased inflammation in the spinal cord. Unlike oral tolerization, spleen cells from i.v. tolerized animals did not suppress adoptively transferred EAE when co-transferred with encephalitogenic cells although i.v. tolerized animals were protected from adoptively transferred EAE. MBP peptides were then utilized to further characterize differences between i.v. and oral tolerization in the actively induced disease model. Both orally and intravenously administered MBP suppressed actively induced EAE. However, EAE was only suppressed by prior i.v. tolerization with the encephalitogenic MBP peptide 71-90, but not with the non-encephalitogenic peptide 21-40, whereas prior tolerization with 21-40 did suppress actively induced EAE when administered orally. These results suggest a different mechanism of tolerance is initiated by oral vs. intravenous administered antigen. Specifically, oral tolerization suppresses primarily by the generation of active suppression whereas the dominant mechanism of suppression associated with i.v. tolerization appears most consistent with the elicitation of clonal anergy.

Effect of oral beta interferon on subsequent immune responsiveness.

Oral administration of myelin antigens reduces the incidence and severity of EAE in rat and mouse models and decreases the frequency of MBP-reactive cells and the frequency of attacks in some patients with multiple sclerosis. Low-dose oral tolerance has been shown to be mediated by Th2-type regulatory cells that secrete TGFbeta and IL-4/IL-10. Adjuvants and cytokines may modulate oral tolerance. The addition of betaIFN to the experimental therapy regimen, either orally or by intraperitoneal injection, has been shown to enhance the suppressive effects of oral myelin antigens when either are fed the suboptimal dosing regimen to suppress EAE. The current studies were conducted to elucidate the mechanism of the observed in vivo synergy of betaIFN and antigen feeding. Analysis of the in vitro proliferative response and cytokine production by lymphocytes from fed animals in response to specific antigen in culture shows that the synergistic effect may be related to both independent suppression of the immune response by oral betaIFN and enhanced production of TGFbeta and IL-4/IL-10. There was an unexpected increase in the production of gammaIFN by lymphocytes in vitro after three doses of oral betaIFN in vivo. These observations have important implications for the use of cytokines to modulate oral tolerance.

Decreased CNS inflammation and absence of clinical exacerbation of disease after six months oral administration of bovine myelin in diseased SJL/J mice with chronic relapsing experimental autoimmune encephalomyelitis

Murine chronic relapsing experimental autoimmune encephalomyelitis (CR‐EAE) is a model of inflammatory demyelinating disease of the central nervous system (CNS) with similarity to multiple sclerosis (MS) in humans. Mice with confirmed neurologic deficits from CR‐EAE were treated by oral administration of whole bovine myelin to investigate the effect of long‐term oral delivery of myelin antigens on clinical disease and on the inflammatory response in the CNS. EAE‐positive mice were fed doses of 1 mg, 10 mg, or 20 mg of bovine myelin every other day for 6 months. We found that prolonged oral delivery of neuroantigen suppressed inflammatory and demyelination foci in the CNS of myelin‐treated mice with no exacerbation of clinical disease status compared with the control group. Analysis of histologic sections of brain and spinal cords with hematoxylin‐eosin (H&E) and Luxol fast blue (LFB) staining showed a decrease in the inflammatory cell infiltration and active centers of demyelination, respectively. Furthermore, after 6 months of treatment, there was no increased sensitization to myelin antigens seen, as measured by antimyelin basic protein (MBP) or anti‐proteolipid apoprotein (PLP) antibodies. These results demonstrate that prolonged oral administration of myelin antigens in diseased animals has an ameliorating effect on the pathologic process and supports its potential long‐term use in humans with MS.

Biological activity of recombinant human myelin basic protein

Using an inducible expression vector in Escherichia coli, we have expressed, purified, and biologically characterized recombinant human myelin basic protein (r-MBP). The recombinant protein binds in cation-exchange chromatography with similar affinity to purified, human MBP, and elutes as a single, 18.5-kDa species as judged by SDS-PAGE. The recombinant protein exhibits similar conformation to native MBP, as demonstrated by ELISA reactivity with a panel of six monoclonal antibodies directed against at least three different epitopes on human MBP. Additionally, recombinant MBP can trigger the proliferation of human T cell clones recognizing MBP, can induce experimental autoimmune encephalomyelitis (EAE) in the SJL mouse, and is capable of suppressing EAE following tolerization via oral administration. Most important, by using a novel method of purification, the recombinant protein preparation contains no detectable proteolytically derived fragments of MBP, a significant advantage over MBP purified from protease-rich central nervous system tissue. Purified recombinant MBP produced in E. coli will be useful as a biological reagent where highly purified protein devoid of degradation products is needed. Relevance to the study of antigen processing, interactions between MHC and the TCR, as well as for the investigation of antigen-driven immune tolerance are discussed.

Suppression of Organ-Specific Autoimmune Diseases by Oral Administration of Autoantigens

One of the primary goals in developing effective therapy for autoimmune diseases is to specifically suppress autoreactive immune processes without affecting the remainder of the immune system. Autoimmune diseases involve the presence of autoreactive clones that have not been deleted in the thymus and thus these cells must be inactivated in the periphery. We have been investigating antigen-driven peripheral immune tolerance as a means to suppress autoimmune processes using the oral route of antigen-exposure to the immune system because of its inherent clinical applicability. An effective andlong-recognized method of inducing immunologic tolerance is the oral administration of antigen, which was first demonstrated by Wells for hen’s egg protein.[l] The mechanism by which orally administered antigen induces tolerance most probably relates to the interaction of protein antigens with gut-associatedlymphoid tissue (GALT) and the subsequent generationo? regulatory or suppressor T cells. [2] The two primary points of contact of orally administered antigen are Peyer’s patches and gut epithelial cells, thelatter of which overlie intraepitheliallymphocytes. Investigators have reported that specific suppressor cells can be found in the Peyer’s patches following oral administration of antigen and that such cells then migrate systemically.[2] Intestinal epithelial cells express class II antigens on their surface and thus have the capacity to function as antigen-presenting cells.[3] Furthermore, it has been shown that human gut epithelial cells preferentially stimulate CD8+ cells in vitro which can function to suppress in vitro immune responses. [4] Although most investigators have reported that the generation of antigen-specific suppressor T cells is the primary mechanism responsible for mediating oral tolerance, other reported mechanisms include anti-idiotypic antibodies, immune complexes and biologically filtered antigen (reviewed in REF. 5).

Rat Experimental Autoimmune Encephalomyelitis

Publisher Summary This chapter explains experimental autoimmune encephalomyelitis (EAE) in the rat. EAE has been the most widely used model for the human disease, multiple sclerosis. EAE may be actively induced by immunization with myelin basic protein (MBP) or proteolipid apoprotein (PLP), or by specific peptides encompassing the encephalitogenic regions. MBP and PLP are the major protein components of central nervous system myelin. For disease induction, the protein or peptide must be emulsified in Freunds complete adjuvant, or adsorbed on the surface of particulates such as carbon, kaolin, or talc. The emulsified adjuvant acts as a slow-releasing repository of emulsified antigen at the injection site and creates a granulomatous reaction that stimulates mononuclear cells to induce a CD4+ T-cell response. Freunds complete adjuvant holds an established place in the laboratory as a means of attaining high levels of antibody against protein antigens and inducing cell-mediated responses.

T cell recognition of immunodominant and cryptic proteolipid protein epitopes in humans

We investigated the immune response to proteolipid protein (PLP), the most abundant central nervous system myelin protein in humans. A total of 8207 short-term T cell lines were generated from 49 individuals, 39 patients with multiple sclerosis and 10 control subjects. As we have reported previously, the frequency of PLP-reactive T cells did not differ between the two groups. To determine immunodominant PLP epitopes, proliferative responses of 971 PLP-specific lines were tested with 27 overlapping 20-amino acid peptides encompassing the human PLP sequence and the binding affinities of the PLP peptides to DRB5*0101 and DRB1*1501, DR2 MHC class II isotypes associated with multiple sclerosis, were determined. The T cell response after primary PLP stimulation was focused on two immunodominant epitopes comprising residues p30-49 and p180-199. These two fragments were recognized after processing of native protein by APCs and were situated in hydrophilic regions of PLP exhibiting only moderate affinity to DR2 molecules. In contrast, when T cells from DR2+ subjects were stimulated initially by individual synthetic peptides with either high or low affinity to DRB5*0101 and DRB1*1501 isotypes, additional cryptic epitopes were recognized. MHC restriction of lines specific for the cryptic PLP epitopes were related to binding affinity to DR2 isotypes. Our results indicate that protein Ags are recognized in vivo as immunodominant epitopes after Ag processing by APCs and as cryptic epitopes after processing, presumably by extracellular proteolytic enzymes.