Exploring the Autoimmune Process in Pediatric Systemic Lupus Erythematosus

Virginia Pascual, M.D.

Baylor Institute for Immunology Research

Funded in September, 2002: $300000 for 4 years
LAY SUMMARY . ABSTRACT . BIOGRAPHY . FINDINGS . SELECTED PUBLICATIONS .

LAY SUMMARY

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Exploring the Autoimmune Process in Pediatric Systemic Lupus Erythematosus

Systemic Lupus Erythematosus (SLE) is an autoimmune disease in which immune cells mistakenly identify the body's own cells as "foreign" and attack them. It produces damage intermittently (an "on-again, off-again" course), affecting multiple organs including the skin, kidneys, and central nervous system. About 25 percent of patients are children; many of them suffer from kidney failure. Lacking an understanding of the specific cause(s), current treatment consists of generalized use of anti-inflammatory and immuno-supressive drugs. This treatment carries many side effects, especially in children.

Baylor Institute scientists' research on the cause of SLE, however, may lead to a better understanding of this and perhaps other autoimmune diseases and to highly targeted therapy. The researchers' recent studies suggest that SLE may be caused by alterations in dendritic cells. These are immune cells that normally recognize a "foreign" invading antigen, and display the antigen's molecular structure to immune T and B cells, which then find and attack the antigen.

When dendritic cells misidentify the patient's own cells as "foreign" and direct T and B cells to attack, however, an autoimmune response occurs. The scientists previously found that pediatric SLE patients have an over-expression of interferon, an immune substance that can prompt immature (undifferentiated) cells in patients' blood to develop several features of dendritic cells.

Using a new approach, termed "global gene expression analysis," to determine which genes are expressed at higher levels in blood cells taken from patients with SLE, Dr. Pascual has found that the overrepresented genes were of two classes: genes that are turned on in response to interferon, and genes that are expressed by young or immature white blood cells called granulocytes.

The researchers hypothesize that the immature granulocytes are contributing to the disease. One of their hypotheses is that the granulocytes are captured by the interferon-activated dendritic cells and serve as "self" antigens that drive the autoimmune attacks in SLE. They will explore this hypothesis by characterizing the undifferentiated (immature) granulocytes, establishing whether these cells infiltrate the skin, kidney and nervous system, and determining whether these cells are responsible for the organ tissue destruction characteristic of SLE. If so, therapies that can control interferon or the immature granulocytes might provide new and highly targeted treatments.

ABSTRACT

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Exploring the Autoimmune Process in Pediatric Systemic Lupus Erythematosus

Systemic Lupus Erythematosus (SLE) is a prototype systemic autoimmune disease characterized by flares of high morbidity for which we have no predictors. SLE pathogenesis remains enigmatic, and therefore current therapies are based on non-specific immuno-suppression. With this is mind, we used oligonucleotide microarrays to analyze the genes expressed by blood mononuclear cells from active SLE patients compared to arthritis patients and healthy controls. We found that SLE can be distinguished by a remarkably homogeneous gene expression pattern with predominant overexpression of granulopoiesis-related and interferon-induced genes. The IFN signature confirmed our earlier findings on the central role of IFN alpha in this disease. The presence of a granulopoiesis signature led us to consider SLE as a disease of the innate immunity. Indeed, enzymatically active immature granulocytes that are abundant in SLE blood, as opposed to healthy blood, could explain the devastating tissue damage (i.e. nephritis and vasculitis) in SLE patients. Thus, immature granulocytes acting as effector cells in SLE could lead to cell death, and therefore could represent a novel target for therapy.

The present proposal is designed to test the hypothesis described above. Three aims are proposed: the first focuses on the characterization of the blood immature neutrophils; the second will establish whether immature neutrophils infiltrate the main target organs, i.e. kidneys, blood vessels, and skin; the third will analyze whether neutrophil-induced cell death may explain tissue damage in SLE.
While our primary focus will be our pediatric SLE population, we will extend this investigation to the adult population. This will permit us to conclude whether adult SLE is driven by the same pathophysiology as pediatric SLE.

INVESTIGATOR BIOGRAPHIES

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Virginia Pascual, M.D.

Dr. Virginia Pascual received her M.D. in 1981 at the Universidad Complutense in Madrid, Spain, and completed a Pediatrics Residency at the Hospital "12 de Octubre," Universidad Complutense in Madrid. From 1988 through 1992 she completed a post-doctoral fellowship in molecular immunology at UT Southwestern Medical Center under the direction of Dr. J. Donald Capra. From 1992 through 1995 she did a clinical fellowship in Pediatric Rheumatology at the same institution under the direction of Dr. Chester W. Fink. In 1995 she was appointed Assistant Professor of Pediatrics at UT Southwestern.

Dr. Pascual is board-certified in Pediatrics and Pediatric Rheumatology and is currently the director of the Pediatric Rheumatology Division at UT Southwestern. Dr. Pascual's laboratory focuses on understanding the pathogenesis of pediatric autoimmune diseases, with especial emphasis on Systemic Lupus Erythematosus (SLE) and Juvenile Arthritis. Her studies so far have led to the identification of dendritic cells, and the cytokines that these cells produce, as important players in the development of SLE. The information gathered from these studies may lead to the design of better, targeted therapies for these patients.

FINDINGS

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Scientific Results:
Gene expression profiling of active Systemic Lupus Erythematosus (SLE) mononuclear cells show a significant granulopoiesis signature that can be traced down to the presence of immature blood neutrophil precursors. Indeed, using antibodies against CD16 and CD11b, neutrophils can be separated into three stages: immature neutrophil (IN) stage I (CD16-/CD11b-); IN stage II (CD16-/CD11b+), and stage III or mature neutrophil (CD16+/CD11b+). While in healthy individuals cells negative for either CD16 or CD11b are not found in the blood, blood neutrophils in SLE patients contain cells belonging to all three stages. We have successfully obtained RNA from highly pure populations of immature (stage I) and mature (stage III) SLE as well as mature healthy blood neutrophils. After RNA amplification, and hybridization to Affymetrix U133 gene chip arrays, we have detected i) genes that are specifically transcribed in immature versus mature neutrophils, ii) neutrophil genes that contribute to the SLE-specific gene signatures. Furthermore, we have found that the presence of immature neutrophils correlates with SLE disease activity and with the development of renal disease, suggesting that they are relevant to disease pathogenesis and severity.

The presence of immature neutrophils in SLE blood could be a reactive process due to the apoptosis of mature cells. We have found that mature SLE neutrophils display accelerated spontaneous apoptosis when compared with healthy mature neutrophils in culture. Despite an increased apoptosis rate, mature SLE neutrophils promptly release as much IL-8 and MIP1-alpha as healthy cells when stimulated with the TLR2 agonist lipopeptide and TLR7/8 agonist R848, implying that these cells are functional. The release of cytokines by pro-apoptotic SLE neutrophils could be contributing to an inflammatory environment, which would facilitate the maturation of antigen-presenting cells and the processing of apoptotic material in an immunogenic manner, explaining some of the key pathogenic events in SLE. We have also found that the number of immature neutrophils in the SLE blood mononuclear fraction correlates with the ability of the patients’s serum to induce apoptosis of healthy mature neutrophils. Anti-neutrophil antibodies and/or death-inducing molecules like TRAIL do not seem to be responsible for the pro-apoptotic effect of SLE serum on healthy neutrophils. We are currently investigating whether SLE serum immune complexes contribute to this pro-apoptotic effect.

SELECTED PUBLICATIONS

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Yurasov S., Tiller T., Tsuiji M., Velinzon K., Pascual V., Wardemann H., and Nussenzweig M.C.  Persistent expression of autoantibodies in SLE patients in remission.   J Exp Med. 2006 Oct 2;203(10):2255-61.

Yurasov S., Wardemann H., Hammersen J., Tsuiji M., Meffre E., Pascual V., and Nussenzweig M.C.   Defective B cell tolerance checkpoints in systemic lupus erythematosus.   J Exp Med. 2005 Mar 7;201(5):703-11.