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Leukocyte adhesion to the endothelial venules in the pancreatic islets is thought to be one of the initial steps in the development of insulin-dependent diabetes mellitus. It has been suggested that leukocyte adhesion to endothelium is a sequential multistep process involving various different homing receptors. We report here that blocking different homing receptors--namely, L-selectin and very late antigen 4 (VLA-4)--which function during different stages of the adhesion process, by specific monoclonal antibodies inhibits insulitis and prevents diabetes in mice. Moreover, leukocyte attachment to the inflamed vessels within pancreatic sections could be inhibited by anti-L-selectin and anti-VLA-4 antibodies. Interestingly, anti-L-selectin or anti-VLA-4 antibody did not appear to influence the autoimmune response to a panel of pancreatic beta-cell autoantigens. These data suggest that L-selectin and VLA-4 receptors are involved in mediating leukocyte homing to the islets and that intervention of these two adhesion pathways may provide a novel approach for treatment of autoimmune diseases such as insulin-dependent diabetes mellitus.

Murine neuroblastoma N1E-115 cells are a useful system in which to study neuronal angiotensin II (AngII) receptors. N1E-115 cells possess both type 1 (AT1) and type 2 (AT2) AngII receptor subtypes, as does mammalian brain. AT2 receptors in brain or N1E-115 cells can be solubilized in 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. In the present study, heparin-Sepharose chromatography was used to partially purify solubilized N1E-115 membranes to produce an enriched population of AT2 receptors. Subsequently, an eluted peak, containing the majority of AT2 binding activity, was used as an immunogen in the development of protein-directed polyclonal antibodies. The antibodies specifically detected immunoreactive proteins of approximately 110 and 66 kDa in both solubilized N1E-115 cells, as well as the original protein material that eluted from the heparin-Sepharose column, whereas no such immunoreactivity was detected in a kidney epithelial cell line that lacks any specific 125I-labeled AngII (125I-AngII) binding activity. Moreover, the antibodies immunoreacted with affinity-purified AT2 receptors. These antibodies were also able to immunoprecipitate AT2 receptors from solubilized N1E-115 cells, as revealed by the pharmacologic profile of 125I-AngII binding to the precipitated protein. Similarly, the antibodies were able to immunoprecipitate a 66-kDa protein that had been covalently crosslinked with 125I-AngII by use of the homobifunctional crosslinker dithiobis(succinimidyl propionate). Collectively, these results demonstrate the development of a specific AT2 receptor antibody that may be used to further characterize this receptor subtype at both the cellular and molecular levels.

To elucidate the role of cell adhesion molecules in the pathogenesis of insulin-dependent diabetes mellitus and to determine the predominant lymphocytic homing pathway(s) involved in the selective lymphocytic infiltration of pancreatic islets (insulitis), nonobese diabetic mice were treated with monoclonal antibodies specific for the L-selectin and integrin alpha 4 lymphocyte adhesion molecules. Treatment of neonatal mice with either anti-L-selectin or anti-integrin alpha 4 monoclonal antibodies for the first 4 weeks of life led to a significant and long-term protection against spontaneous occurrence of insulitis and diabetes. The same treatment failed to inhibit lymphocytic infiltration of the salivary glands (sialadenitis). This tissue-specific inhibition of inflammation may be attributed to differences between the pancreas and salivary gland in their expression of endothelial ligands for L-selectin (peripheral vascular addressin) and for integrin alpha 4 (mucosal addressin cell adhesion molecule 1 and vascular cell adhesion molecule 1). Mucosal addressin cell adhesion molecule 1 is highly expressed by vessels within the inflamed islets but was not detected in the salivary glands. In contrast, peripheral vascular addressin- and vascular cell adhesion molecule 1-expressing vessels can be found in almost every area of inflammation within the salivary glands but are seen in only 40-50% of inflamed islets. Anti-L-selectin and anti-integrin alpha 4 treatment had no demonstrable effect on anti-beta-cell autoimmunity or on the immune responses to foreign antigens. Therapeutic treatment with anti-L-selectin after the onset of insulitis from 10 to 14 weeks of age delayed the onset but failed to prevent spontaneous insulin-dependent diabetes mellitus, whereas anti-integrin alpha 4 treatment resulted in a significant and long-lasting suppression of the disease. These data strongly suggest that integrin alpha 4 plays a prominent role in the spontaneous development of insulitis and diabetes in nonobese diabetic mice.

Susceptibility to the human autoimmune disease insulin-dependent diabetes mellitus is strongly associated with particular haplotypes of the major histocompatibility complex (MHC). Similarly, in a spontaneous animal model of this disease, the nonobese diabetic (NOD) mouse, the genes of the MHC play an important role in the development of diabetes. We have produced transgenic NOD mice that express the class II MHC molecule I-Ad in addition to the endogenous I-Ag7 molecules in order to study the role of these molecules in the disease process. Although the inflammatory lesions within the islets of Langerhans in the pancreas appear similar in transgenic and nontransgenic animals, transgenic mice develop diabetes with greatly diminished frequency compared to their nontransgenic littermates (10% of transgenic females by 30 weeks of age compared to 45% of nontransgenic females). Furthermore, adoptive transfer experiments show that T cells present in the transgenic mice are able to interfere with the diabetogenic process caused by T cells from nontransgenic mice. Thus, the mechanism by which I-Ad molecules protect mice from diabetes includes selecting in the thymus and/or inducing in the periphery T cells capable of inhibiting diabetes development.

Long-term depression (LTD) of glutamatergic and electrotonic transmission can be induced at mixed synapses between eighth nerve fibers and the goldfish Mauthner (M) cell in vivo, by pairing weak presynaptic tetani with postsynaptic inhibition. This LTD can be reversed by stronger tetani that produce long-term potentiation (LTP). Moreover, the depression is more likely to occur and tends to last longer when the initial synaptic efficacy is high--that is, if the synaptic strength is first potentiated. In addition, when synaptic efficacy is initially elevated, a weak tetanization that usually results in a gradually developing potentiation instead produces no change in chemical transmission and even a depression of electrotonic coupling. Thus, the modifications in synaptic transmission caused by a certain tetanizing protocol depend upon the history of synaptic efficacy. This last concept provides an experimental basis for theoretical models concerned with pre- and postsynaptic contributions to the regulation of synaptic plasticity.