The intestine is the largest lymphoid organ in the body, challenged constantly by an enonnous quantity and diversity of antigens. Distinct from peripheral lymphocytes, intestinal lymphocytes have evolved unique mechanisms of tolerance and appear to govern mucosal processes such as "chronic physiologic inflammation" and oral tolerance. Failure of mucosal tolerance has been implicated in the pathogenesis of several diseases, including inflammatory bowel disease, celiac disease, and even autoimmune diabetes. One population of intestinal lymphocytes, intraepithelial lymphocytes (IELs), exists within the intestinal epithelium itself and remains poorly characterized. IELs respond to unique activation signals and appear to be in part responsible for the maintenance of epithelial integrity and mucosal tolerance. Type 1 diabetes is one of the most common chronic childhood illnesses and causes significant morbidity and mortality. Type 1 diabetes mellitus is an autoimmune disease that results from immune-mediated destruction of insulin-producing pancreatic beta cells and is characterized by an absolute insulin deficiency. Several animal models are used to study the immunopathogenesis of type 1 diabetes, including the BB rat and NOD mouse. BBDP rats spontaneously develop autoimmune diabetes mellitus and are severely deficient in peripheral T cells. BBDR rats do not spontaneously develop autoimmune diabetes, have nonnal numbers of peripheral T cells, and can be induced to become diabetic by injections of a cytotoxic anti-ART2a mAb and low doses of poly I:C. The cause of autoimmune diabetes in BB rats and humans is still unknown, but both genetic and environmental factors appear to participate. I hypothesize that one important class of environmental factors--diet and enteromicrobial agents--participates in this pathogenic process through the mediation of the gut immune system. In this dissertation, I report a new method for the isolation of rat IELs that is based on the selective removal of intestinal epithelial cells under conditions that leave the basement membrane undisturbed. The yield of rat IELs using this method is 5-10 fold greater than that reported for other methods. Morphological and phenotypic analyses demonstrate that the purified cell population is comprised of IELs and is not contaminated with lamina propria or Peyer's patch lymphocytes. Phenotypic analysis reveals 5 major subsets of IELs, including populations of γδ T and natural killer (NK) cells present at levels not previously detected. I also report that rat intraepithelial NK (IENK) and peripheral NK cells are similar in morphology, in their ability to lyse NK-sensitive targets, and in their ability to suppress a one-way mixed lymphocyte culture. In contrast, IENK cells differ from splenic NK cells phenotypically, and a substantial fraction of IENK cells appear to spontaneously secrete IL-4 and/or IFN-γ. I conclude that rat IELs harbor a large population of NKR-P1A+ CD3-cells that function as NK cells but display an activated phenotype and unusual cytokine profile that clearly distinguish them from splenic NK cells. Their phenotypic and functional characteristics suggest that these distinctive intraepithelial NK cells may participate in the regulation of mucosal immunity. I next demonstrate that, prior to diabetes, both BBDP and ART2a-depleted BBDR rats have a reduced total number of IELs and exhibit a selective deficiency of IENK cell number and function as compared to control BBDR rats. The deficiency of BBDP rat IELs can be corrected by engraftment of bone marrow from histocompatible WF donors. These results suggest 1) that the peripheral lymphopenia in BBDP rats extends to the IEL compartment, particularly to IENK cells, 2) that in BBDR rats the diabetes-inducing treatment depletes IELs, particularly IENK cells, and 3) that the defect in BBDP rat IELs is intrinsic to hematopoietic cells, not intestinal stromal cells. I also establish that, unlike BBDR and WF rats, BBDP rats are also deficient in γδTCR+IELs, a population of T cells that may play a role in normal mucosal tolerance. In addition, I report preliminary data supporting the hypothesis that systemic autoreactivity may be initiated in the intestine; peripheral autoreactive lymphocyte populations appear to emanate first from mesenteric lymph nodes that drain the intestine, and such cells may initiate a type 2 autoimmune phenomenon driven by IL-4. Collectively, my findings support the hypothesis that a failure of mucosal tolerance in BBDP rats, perhaps secondary to deficiencies in one or more IEL subpopulations, participates in the pathogenesis of autoimmune diabetes in these animals by activating peripheral autoreactive T cells. The nature of the autoimmune response in BB rats (driven by IL-4) appears to be distinct from that of NOD mice. Despite the differences between these two well-accepted animal models of autoimmune diabetes, until more is known about the pathogenesis of type 1 DM in humans, lessons learned from both the BB rat and NOD mouse continue to be of tremendous benefit to our understanding of human disease.

The glycophosphatidylinositol(GPI)-linked membrane protein ART2 is an antigenic determinant for T lymphocytes that regulate the expression of diabetes in the BB/W rat model. Though little is understood of the physiologic role of ART2 on T lymphocytes, ART2 is a member of the mono-ADP-ribosyl transferase subgroup ofthe ADP-ribosyl transferase (ART) protein family. The ART protein family, which traditionally has been divided into mono-ADP-ribosyl transferases (mono-ARTs), poly(ADP)-ribose polymerases (PARPs), and ADP-ribosyl cyclases, influences various aspects of cellular physiology including: apoptosis, DNA damage repair, chromatin remodeling, telomere replication, cellular transport, immune regulation, neuronal function, and bacterial virulence. A structural alignment of ART2.2 with chicken PARP indicated the potential for ART2.2 to catalyze ADP-ribose polymers in an activity thought to be specific to the PARP subgroup and important for their regulation of nuclear processes. Kinetic studies determined that the auto-ADP-ribosyl transferase activity of ART2.2 is multitmeric and heterogeneous in nature. Hydroxylamine-cleaved ADP-ribose moieties from the ART2.2 multimers ran as polymers on a modified sequencing gel, and digestion of the polymers with snake-venom phosphodiesterase produced AMP and the poly(ADP)ribose-specific product, PR-AMP, which was resolved by analytical HPLC and structurally confirmed by ESI-MS. The ratio of AMP to PR-AMP was higher than that of PARP raising the possibility that the ART2.2 polymers had a different branching structure than those of PARP. This alternative branching was confirmed by the presence of ribose phosphate polymers in the snake venom phophodiesterase treated samples. The site of the auto-poly(ADP)-ribose modification was determined to be R185, a residue previously proposed to influence the level of auto-ADP ribosylation of ART2.2 by mutational analysis. These data provide the first demonstration of a hybrid between mono-ARTs and PARPs and are the earliest indication that PARP-like enzymes can exist outside the nucleus and on the cell surface.