Biology and therapeutic potential of cannabinoid CB2 receptor inverse agonists.

Evidence has emerged suggesting a role for the cannabinoid CB2 receptor in immune cell motility. This provides a rationale for a novel and generalized immunoregulatory role for cannabinoid CB2 receptor-specific compounds. In support of this possibility, we will review the biology of a class of cannabinoid CB2 receptor-specific inverse agonist, the triaryl bis-sulfones. We will show that one candidate, Sch.414319, is potent and selective for the cannabinoid CB2 receptor, based on profiling studies using biochemical assays for 45 enzymes and 80 G-protein coupled receptors and ion channels. We will describe initial mechanistic studies using this optimized triaryl bis-sulfone, showing that the compound exerts a broad effect on cellular protein phosphorylations in human monocytes. This profile includes the down regulation of a required phosphorylation of the monocyte-specific actin bundling protein L-plastin. We suggest that this observation may provide a mechanism for the observed activity of Sch.414319 in vivo. Our continued analysis of the in vivo efficacy of this compound in diverse disease models shows that Sch.414319 is a potent modulator of immune cell mobility in vivo, can modulate bone damage in antigen-induced mono-articular arthritis in the rat, and is uniquely potent at blocking experimental autoimmune encephalomyelitis in the rat.

The pathogenesis of adoptive murine autoimmune diabetes requires an interaction between alpha 4-integrins and vascular cell adhesion molecule-1.

An adoptive transfer model of insulin-dependent diabetes mellitus (IDDM) in the nonobese diabetic mouse was used to examine the roles of alpha 4-integrin, vascular cell adhesion molecule 1 (VCAM-1); and intercellular adhesion molecule 1 (ICAM-1) in the pathogenesis of autoimmune diabetes. Antibodies specific for both alpha 4-integrin and one of its ligands, VCAM-1, were able to delay onset of diabetes and decrease the incidence of the disease in adoptive transfer studies. This blocking of disease was accompanied by a marked decrease in lymphocytic infiltration of the islets of Langerhans. Furthermore, these antibodies preferentially block entrance of CD4 T cells into the tissue. Antibodies specific for ICAM-1 had little effect on the onset or incidence of IDDM. Thus, we conclude that an alpha 4-integrin-VCAM-1 interaction is important in T cell entry into the islets of Langerhans and in the pathogenesis of IDDM. In addition, the cascade of events leading to T cell transit across endothelium may be different for CD4 and CD8 cells, and may differ depending on the endothelium involved. Our results support the more general conclusion that an alpha 4-integrin-VCAM-1 interaction may be crucial in allowing activated effector CD4T cells to leave the blood and enter tissue to clear infection.

Prevention of diabetes in NOD mice by injection of autoreactive T-lymphocytes.

The nonobese diabetic (NOD) mouse develops a high incidence of autoimmune diabetes and is believed to be a good model for insulin-dependent diabetes mellitus (IDDM) in humans. We isolated T-lymphocyte lines from islets of newly diabetic NOD mice, some of which are autoreactive to NOD spleen cells. Because autoreactive T-lymphocytes have been implicated in immune suppression, we injected NOD mice with an autoreactive T-lymphocyte line. The injected mice had a marked decrease in incidence of IDDM compared with control mice. Moreover, their islets showed no insulitis at 1 yr of age. We conclude that autoreactive T-lymphocytes can prevent the development of IDDM in NOD mice. This result suggests that 1) islets contain both effector cells capable of damaging pancreatic beta-cells and cells able to regulate this autoimmune response, and 2) development of IDDM depends on the balance between these opposing forces.

Expression of the co-stimulator molecule B7-1 in pancreatic beta-cells accelerates diabetes in the NOD mouse.

B7-1 is a co-stimulatory molecule that signals T-cells that recognize antigen to proliferate and differentiate into effector T-cells. The same cell must present antigen and express co-stimulatory molecules, such as B7-1, to activate naive T-cells. Thus, tissues that do not express co-stimulatory molecules would not be expected to induce immune responses, while expression of a co-stimulator on tissue cells may convert them into effective antigen-presenting cells and induce autoimmunity. To test this, transgenic mice have been generated that express B7-1 on the beta-cells of the pancreatic islets of Langerhans. On a B6 genetic background, B7-1 expression on beta-cells does not predispose to diabetes. B6 mice are resistant to diabetes. However, when B7-1 is expressed on the beta-cells of B6 mice backcrossed once to the genetically susceptible NOD strain, the onset of diabetes is accelerated and the autoimmune attack intensified. This illustrates that B7-1 is a very potent co-stimulatory molecule in vivo and that its presence on the surface of tissue cells can potentiate the autoimmune process.

Treatment with sulfatide or its precursor, galactosylceramide, prevents diabetes in NOD mice.

Sulfatide (3'sulfogalactosylceramide) is a glycosphingolipid present within the nervous system and in the islets of Langerhans. Anti-sulfatide antibodies have been observed in both pre-diabetic and newly diagnosed type 1 diabetic patients. The aim of this study was to test in vivo, the therapeutic effect of sulfatide on the development of diabetes in the NOD mouse. In four separate experiments diabetogenic splenocytes from newly diabetic NOD mice were injected iv into 7-8 week old irradiated (700R) female NOD mice (4-10 million cells/mouse). Each experiment consisted of four treatment groups to which the mice were randomly divided: 1) sulfatide; 2) galactosylceramide (the precursor to sulfatide without sulfate); 3) GM1, a glycosphingolipid negatively charged as sulfatide but with a different sugar composition; and 4) phosphate buffered saline (PBS). The mice received 100 microg glycosphingolipid iv on the day of cell transfer and 1-3 times thereafter at four day intervals, and were screened for diabetes three times a week the next 52 days. Among all the 35 sulfatide-treated mice 54% became diabetic compared to 93 % of 43 PBS-treated animals (p < 0.00001). Correspondingly, galactosylceramide reduced diabetes incidence to 52% (25 mice, p < 0.00001). On the other hand, 86% of GM1-treated mice (n=28) became diabetic indicating that no effect was obtained by this glycosphingolipid. In two experiments in which less spleen cells were transferred (4-5 mill.) and glycosphingolipids were given 4 times, 35% of the sulfatide-treated animals (n = 17) developed diabetes compared to 85% of PBS-treated mice (n = 20, p < 0.001). A robust proliferative response to sulfatide, but none to GM1, was observed when spleen cells were rechallenged with glycosphingolipid in vitro. Thus, like insulin and GAD, sulfatide is able to prevent diabetes in NOD mice.

Type 1 diabetes mellitus: an imbalance between effector and regulatory T cells?

Abundant evidence now exists that autoimmunity plays a critical role in the pathogenesis of type 1 (insulin-dependent) diabetes mellitus. The non-obese diabetic (NOD) mouse is an extensively studied animal model of this T-cell-mediated autoimmune disease. Our laboratory has focused on isolating diabetogenic T cell clones from NOD mice as a means of elucidating the pathogenesis of type 1 diabetes. This experimental approach presupposes that type 1 diabetes in NOD mice results from the action of islet-reactive T cells that are not present in other mouse strains; the diabetogenic T cells would therefore represent "forbidden clones" which exist in NOD mice as a result of a failure of clonal deletion. While the inappropriate presence of diabetogenic T cells probably plays a central role in murine diabetes, it cannot explain all aspects of the disease. Type 1 diabetes is a chronic disorder with a lengthy preclinical stage; if the diabetogenic T cells acted in an unopposed fashion, one might expect to see a much more fulminant clinical course. This observation suggests that regulatory influences are likely to exist in this disease--a possibility supported by recent experimental data. If these regulatory influences could be identified and enhanced, specific immunotherapy for type 1 diabetes could be achieved.

Synthesis and kinetics of cyclization of MHC class II-derived cyclic peptide vaccine for diabetes.

Conformationally constrained cyclic peptides are known to be better vaccines because of their ability to mimic the native structure of a protein against which an immune response is sought. To test the hypothesis of using conformationally constrained, disease-associated, MHC-derived peptides as vaccines for the prevention of type I diabetes, a 22 amino acid nonobese diabetic(NOD) mouse MHC class II-derived synthetic peptide was cyclized by the formation of end-to-end disulfide bonds and used to prevent diabetes and insulitis in NOD mice. The peptide was synthesized by Fmoc chemistry and cyclized using two methods: a commercially available cyclizing resin (Ekathiox) and air oxidation. When a 10 m excess of resin was used, the Ekathiox yielded a substantial amount of cyclic peptide with few or no side reactions. The kinetics of cyclization by air oxidation at different temperatures indicated that increasing both temperature and pH decreased the cyclization time significantly. Air oxidation at pH 10 at 37-55 degrees C yielded the desired product within 2 h.

An explanation for the protective effect of the MHC class II I-E molecule in murine diabetes.

Insulin-dependent diabetes mellitus is widely believed to be an autoimmune disease. Recent onset diabetics show destruction of insulin-secreting pancreatic beta-cells associated with a lymphocytic infiltrate (insulitis), with autoantibodies to beta-cells being found even before the onset of symptoms. Susceptibility to the disease is strongly influenced by major histocompatibility complex (MHC) class II polymorphism in both man and experimental animal models such as the non-obese diabetic (NOD) mouse. As MHC class II molecules are usually associated with dominant immune responsiveness, it was surprising that introduction of a transgenic class II molecule, I-E, protected NOD mice from insulitis and diabetes. This could be explained by a change either in the target tissue or in the T cells presumed to be involved in beta-cell destruction. Recently, several studies have shown that I-E molecules are associated with ontogenetic deletion of T cells bearing antigen/MHC receptors encoded in part by certain T-cell receptor V beta gene segments. To determine the mechanism of the protective effect of I-E, we have produced cloned CD4+ and CD8+ T-cell lines from islets of recently diabetic NOD mice. These cloned lines are islet-specific and pathogenic in both I-E- and I-E+ mice. Both CD4+ and CD8+ cloned T cells bear receptors encoded by a V beta 5 gene segment, known to be deleted during development in I-E expressing mice. Our data provide, therefore, an explanation for the puzzling effect of I-E on susceptibility to diabetes in NOD mice.

Self peptides isolated from MHC glycoproteins of non-obese diabetic mice.

The non-obese diabetic (NOD) mouse spontaneously develops an insulin-dependent diabetes mellitus that resembles human type I diabetes. This disease can be transferred by purified T cells or cloned T cell lines, implicating an autoimmune T cell attack on the pancreatic beta cells of the islets of Langerhans. As all T cell responses involve recognition of peptides bound to MHC molecules displayed at the cell surface, we have examined self peptides binding to the MHC molecules on spleen cells of the NOD mouse. Peptides eluted from the MHC class I molecule Kd have sequences that conform to known motifs for peptides binding this molecule in other strains of mice. The NOD mouse expresses the unique MHC class II molecule I-Ag7. Peptides eluted from I-Ag7 have sequences that implicate an acidic residue in the C terminus of the peptide as important for binding. The role of this residue in binding has been confirmed by direct peptide-binding analysis. This C-terminal acidic amino acid may interact with an arginine residue in the MHC class II alpha-chain that is exposed when beta-chain residue 57 is mutated to serine, or to the unique beta-chain residue histidine 56. These data may provide valuable insights into the nature of autoantigenic peptides presented by NOD mouse MHC molecules by defining the nature of I-Ag7-peptide binding.

Regulatory and effector CD4 T cells in nonobese diabetic mice recognize overlapping determinants on glutamic acid decarboxylase and use distinct V beta genes.

The 524--543 region of glutamic acid decarboxylase (GAD65), GAD65(524--543), is one of the first fragments of this islet Ag to induce proliferative T cell responses in the nonobese diabetic (NOD) mouse model of spontaneous autoimmune diabetes. Furthermore, NOD mice given tolerogenic doses of GAD65(524--543) are protected from spontaneous and cyclophosphamide-induced diabetes. In this study, we report that there are at least two I-A(g7)-restricted determinants present in the GAD65(524--543) sequence, each capable of recruiting unique T cell repertoires characterized by distinct TCR V beta gene use. CD4(+) T cells arise spontaneously in young NOD mice to an apparently dominant determinant found within the GAD65 peptide 530--543 (p530); however, T cells to the overlapping determinant 524-538 (p524) dominate the response only after immunization with GAD65(524--543). All p530-responsive T cells used the V beta 4 gene, whereas the V beta 12 gene is preferentially used to encode the TCR of p524-responsive T cell populations. T cell clones and hybridomas from both of these T cell groups were responsive to APC pulsed with GAD65(524--543) or whole rGAD65. p524-reactive cells appeared to be regulatory upon adoptive transfer into young NOD mice and could inhibit insulin-dependent diabetes mellitus development, although they were unable to produce IL-4, IL-10, or TGF beta upon antigenic challenge. Furthermore, we found that i.p. injection with p524/IFA was very effective in providing protection from cyclophosphamide-induced insulin-dependent diabetes mellitus. These data demonstrate that the regulatory T cells elicited by immunizing with GAD65(524--543) are unique and distinct from those that arise from spontaneous endogenous priming, and that T cells to this limited region of GAD65 may be either regulatory or pathogenic.

Autocrine growth of CD4+ T cells. Differential effects of IL-1 on helper and inflammatory T cells.

The growth factor requirements of cloned lines representing two major subsets of CD4+ T cells were examined. The helper subset, which produces IL-4 as its autocrine growth factor, proliferates in response to IL-2 or to IL-4 in the presence of IL-1. The inflammatory subset, which produces IL-2 as its autocrine growth factor, proliferates in response to IL-2 and, in the presence of limiting amounts of IL-2, shows increased proliferation in the presence of IL-4. The inflammatory subset does not proliferate in response to IL-1 plus IL-4. This ability to respond to the combination of IL-1 plus IL-4 correlates with the presence of IL-1R on the cloned lines tested. These data suggest that IL-1 may play a controlling role in the clonal expansion of CD4+ T cells of different functional types. This, in turn, suggests means by which the immune response could be directed into humoral or cell-mediated responses.

A conformationally-constrained MHC class II I-Ag7-derived peptide protects NOD mice from the development of diabetes.

Allele-specific peptide vaccination against disease-associated MHC class II molecules is a promising new strategy for modulating self-antigen presentation to autoreactive T cells in autoimmune diseases. To evaluate the potential of this approach for treatment of insulin-dependent diabetes mellitus (IDDM), we have designed a cyclic peptide vaccine, DiavaX, from the third hypervariable region of the beta-chain of the NOD mouse MHC class II I-Ag7. NOD mice were treated at 5 and 9 weeks of age with 100 microg DiavaX emulsified in alum, a control peptide in alum, or alum alone. At the end of the study, 87% of alum treated mice had developed diabetes, compared with only 28% of DiavaX-treated mice. None of the control peptides, including a linear I-Ag7, a scrambled cyclic I-Ag7, or an analogous cyclic I-Aspeptide, reduced the incidence of diabetes, demonstrating that the protective effect of DiavaX is conformationally dependent and both allele- and sequence-specific. DiavaX treatment did not cause any general immune suppression, but did induce peptide-specific antibodies and memory T cells. DiavaX-induced protection from diabetes was associated with the maintenance of a non-destructive islet-associated autoimmune response. These data indicate that a conformationally constrained peptide from the disease-associated MHC represents a potential vaccine candidate for the prevention of clinical IDDM.

Cross-linking and conformational change in T-cell receptors: role in activation and in repertoire selection.

TCRs undergo a series of interactions with ligands during development. We have characterized the interaction of a TCR with its ligand and the attendant co-receptor and co-ligand structures. This characterization has led to the model in which the TCR not only binds to class II MHC, but also binds to CD4 co-receptors and co-ligands such as Mls. We have shown that both cross-linking and conformational change in the TCR are required for optimal T-cell activation. Finally, we have used the observation that a particular self-peptide found abundantly associated with class II MHC in the periphery is essentially lacking from thymic cortical epithelium to argue that positive selection for self-MHC recognition may occur by a novel process in the thymic cortex. A TCR recognizing class II MHC with low affinity could either be multiply cross-linked in the absence of conformational change, which here would be driven by a unique peptide, or could be conformationally changed without cross-linking due to the rarity of the individual high-affinity peptide on thymic cortical epithelial cells. Either proposal leads to a partial signal one delivered via the TCR, which we refer to as signal one-half. This signal one-half would induce the cell to repress its other co-receptor molecule and to undergo maturation events such as up-regulation in TCR expression. Such cells are then rigorously screened for activating interactions with autologous structures, such as Mls. The threshold for clonal deletion is set very low to avoid autoreactivity. By this combination of signaling events, a mature TCR repertoire is generated that has the functional characteristics observed in immune systems.