Arthritis & Rheumatism, Volume 62,
November 2010 Abstract Supplement
Abstracts of the American College of
Rheumatology/Association of Rheumatology Health Professionals
Annual Scientific Meeting
Atlanta, Georgia November 6-11, 2010.
Nuclear Modifications during Apoptosis Are Required To Maintain Tolerance to Nuclear Antigens.
Jog1, Neelakshi R., Luning-Prak2, Eline, Caricchio1, Roberto
Our previous data show that in spontaneous models of lupus, the absence of caspase activated DNase (CAD)-generated nuclear modifications during apoptosis results in increased anti-nuclear antibody generation and severe nephritis. These data suggested that CAD generated apoptotic nuclei are a source of auto-antigens (autoAgs) that are involved in negative regulation of autoreactive B cell during development. In the present study we investigated the role of CAD in induction of tolerance in vivo. To this end we used 3H9 mice, an established model of anergy towards chromatin.
3H9 mice carry a rearranged heavy chain reactive against dsDNA, and shows developmental arrest of autoreactive B cells. To determine whether absence of CAD, and therefore autoAgs, breaks tolerance in this anergic model, we generated CAD deficient 3H9 mice [3H9(-/-)]
Contrary to 3H9+/+, 3H9-/- mice were able to break tolerance early in life and produce high titers of anti-dsDNA and anti-chromatin antibodies. The antibodies made in 3H9-/- mice did not, however, differ in specificities as determined by immunofluorescent ANA. The autoreactive B cells in 3H9+/+ mice undergo stringent negative selection due to the presence of autoreactive heavy chain. Therefore we analyzed B cell development and maturation by flow cytometry. The developing B cells from bone marrow were divided into Hardy fractions D (L chain rearrangement), E (immature IgM+), and F (IgM+, recirculating B cells). Compared to wild type, both 3H9+/+ and 3H9-/- mice had increased numbers of fraction D cells, suggesting increased light chain rearrangements. In the periphery, both 3H9+/+ and 3H9-/- mice showed increased mature marginal zone B cells, and immature T2 transitional B cells, and increased lambda inclusion. These data show that absence of CAD does not alter the unique B cell development or maturation in 3H9 mice. The 3H9 heavy chain pairs with endogenous lamda1 light chain to generate anti-dsDNA antibody allowing to follow in vivo the fate of autoreactive B cells. In 3H9 mice lambda1+ cells are excluded from B cell follicles in secondary lymphoid organs and are not able to mature into autoantibody producing B cells. We therefore investigated the fate of lambda1 + cells in the absence of apoptotic nuclear modifications. We stained frozen spleen sections from 3H9+/+ and 3H9-/- mice with B220 to identify B cell follicles and lambda1 to identify the auto-reactive B cells. As expected, in 3H9+/+ mice the lambda1+ cells did not enter the B cell zone and accumulated at the T-B interface. In 3H9-/- mice, however, lambda1+ cells were not excluded from B cell follicles, and entered the B cell zone. These data show that in agreement with our hypothesis, auto-reactive B cells escape to the periphery in absence of CAD-dependent nuclear fragmentation.
Based on our data we conclude that in mice genetically predisposed to autoimmunity, the apoptotic nuclear modifications generated by CAD are necessary to maintain tolerance toward nuclear Ags.
To cite this abstract, please use the following information:
Jog, Neelakshi R., Luning-Prak, Eline, Caricchio, Roberto; Nuclear Modifications during Apoptosis Are Required To Maintain Tolerance to Nuclear Antigens. [abstract]. Arthritis Rheum 2010;62 Suppl 10 :439