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Abstract

Grant Number:	1Z01AR041137-02
Project Title:	Animal Models of Autoimmunity

PI Information:	Name	Email	Title
	LIPSKY, PETER E.	peter.lipsky@email.swmed.edu	SCIENTIFIC DIRECTOR

Abstract: The Animal Models of Autoimmunity Group has made exciting progress over the past year in understanding the role of both tumor necrosis factor (TNF) and IL-21 in the immunoregulation of B lymphocytes. TNF neutralization in patients with rheumatoid arthritis has demonstrated efficacy in improving the signs and symptoms of the disease. This includes lowering of rheumatoid factor titers. However, TNF blockade results in the development of anti-nuclear autoantibodies (ANA) and double-stranded DNA antibodies in a portion of the treated patients. Thus, while TNF neutralization inhibits autoantibody production to one self antigen (immunoglobulin), it promotes B cell reactivity to other autoantigens, such as nuclear proteins and DNA. Last year we showed that mice that are deficient in TNF develop anti-nuclear autoantibodies (ANA), but very rarely develop kidney disease. My students, Rachel Robbins and Carolyn Karafiath found that although loss of TNF drove autoantibody production that deposited in kidneys, no lymphocytic inflammation occured due to the loss of TNF (which is a pro-inflammatory molecule). In the spleen, whereas naive B cells in TNF deficient mice localized correctly to the outer PALS, PNA+ B cells inappropriately clustered within the inner T cell areas of the splenic white pulp. We found that CD4+ T cells isolated from TNF-deficient animals displayed an age-related prominent upregulation of activation markers, which appeared with kinetics similar to ANA production. Moreover, autoantibody was not detected in T-cell/TNF-deficient mice, suggesting that the primary defect driving autoantibody production is inappropriate T cell activation. These data have important implications in the treatment of rheumatic diseases with drugs that inhibit TNF?s actions. This year, over her Christmas vacation, Rachel Robbins went on to show that in T cell transfer studies, OVA-specific OT-2 transgenic T cells alone were not able to confer autoimmunity in TNF/T cell-deficient mice. However, when these mice were immunized with specific antigen, and TNF was neutralized, high titers of ANA rapidly developed. These data suggest that in TNF-deficient mice, activated autoreactive B cells become trapped within the splenic T cell areas and are induced to secrete autoantibodies via CD4+ T cell help provided by activated bystander T cells. TNF, therefore, appears to play a role in maintaining tolerance of both T and B cells, the loss of which results in autoimmunity. Recently, we have turned our attention to a newly discovered cytokine, termed ?IL-21?. This protein is secreted only by activated CD4 T cells, and known to be important in T cell and NK cell activity. Although many molecules have been identified that are important in B cell maturation, the precise signals that induce naive human B cells to undergo class switch recombination and plasma cell differentiation optimally are unknown. We have now demonstrated that IL-21 is a powerful stimulus of human peripheral blood and tonsilar B cells with the capability to drive 100% of poorly responsive naive cord blood B cells to terminally differentiated plasma cells! When highly purified naive B cells were stimulated with anti-CD40, IL-21 dramatically increased proliferation and plasma cell differentiation as well as robust immunoglobulin secretion. In marked contrast, when B cells were stimulated with anti-IgM, the addition of IL-21 resulted in death of activated proliferating naive B cells. However, when B cells were activated with both CD40 and IgM antibodies in the presence of IL-21, rapid differentiation into IgG secreting plasma cells was observed. Yong Sing da Silva showed that these antibodies were predominantly of the IgG1 and IgG3 subclass, which won him an award at our annual NIAMS retreat for best poster presentation. Anna-Marie Fairhurst in our group demonstrated thatt consistent with plasma cell differentiation, BLIMP-1 and AID mRNAs were induced by IL-21. However, Gary Sims determined that the Ig heavy chain genes remained unmutated. Importantly, IL-21 with no additional in vitro stimulation, directly induced CD27+ memory B cells to differentiate and secrete immunoglobulin. In all circumstances, neither IL-2 nor IL-4 induced significant B cell differentiation although IL-2 enhanced, whereas IL-4 inhibited the actions of IL-21. These results indicate that IL-21 plays a critical role in determining the outcome of B cell stimulation, with the potential to foster the differentiation of large numbers of immunoglobulin secreting plasma cells. Rachel Robbins will now turn her attention to the subcellular mediators that IL-21 is inducing to better understand how IL-21 is driving such potent B cells signals. To achieve this goal, RNA will be isolated from IL-21-stimulated human cord, peripheral and tonsilar B cells under different conditions. She will then use gene chip technology to discern what genes are modulated following exposure to IL-21. Real Time PCR will then be used to verify these results. Furthermore, in the near future we will determine if IL-21 is involved in the autoimmune process by studying how it may be involved in human systemic lupus erythematosus and Rheumatoid arthritis. Our group is dedicated to understanding how cytokines are contributing to the development of systemic lupus erythematosus and rheumatic diseases. We feel that our research will lead to new therapeutics as well as to understanding the mechanisms of basic B cell biology.

Public Health Relevance:
This Public Health Relevance is not available.

Thesaurus Terms:
autoimmunity, cytokine, rheumatoid arthritis, systemic lupus erythematosus
B lymphocyte, autoantibody, disease /disorder model, immunoregulation, tumor necrosis factor alpha
genetically modified animal, human subject, laboratory mouse, microarray technology, polymerase chain reaction

Institution:
Fiscal Year:	2004
Department:
Project Start:
Project End:
ICD:	NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
IRG:	AB

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