Hypothesis: In systemic lupus erythematosus (SLE) differentiation and maturation of myeloid dendritic cells (My-DC) are impaired and play an important role in the development of abnormal T cell (TC) responses that contribute to the pathogenesis of the disease. Background and rationale: DCs are key regulators of the immune system and potent TC stimulators. It has been proposed that systemic autoimmune responses that characterize SLE could be explained by alterations in DC function. However, information is needed regarding whether DCs from SLE patients have abnormal differentiation, maturation and abnormal interactions with autologous TCs and the role that these abnormalities could play in the pathogensis of SLE.
Specific aims : 1. Compare phenotypic and functional parameters in monocyte derived DCs from SLE patients and controls 2. Determine the specific characteristics of TC-DC interactions in SLE patients when compared to controls, including cell proliferation, differentiation/maturation and death. Methods: A) DC maturation and differentiation in response to specific stimuli will be evaluated in SLE patients and healthy controls by flow cytometry and multiplex RT-PCR. DC sensitivity to different apoptotic stimuli will be compared in both groups with Annexin V/Propidium iodide staining. B) Capacity of SLE and control DCs to phagocytose and internalize apoptotic and necrotic cells will be evaluated by DC incorporation of CMFDA-labeled apoptotic or necrotic cells using flow cytometry and fluorescent microscopy. Phagocytic capacity will also be evaluated using FITC-dextran. C) Cytokine production by SLE and control monocyte-derived DCs will be determined using multiplex ELISA and results confirmed by RT-PCR. D) DC-TC interactions will be addressed by determining apoptosis susceptibility in co-culture of both cell types and T cell subsets using flow cytometry, cytotoxicity assays and inhibition assays with specific monoclonal antibodies. E) The role of autologous DCs in inducing T cell proliferation and activation of specific TC subsets in SEE patients will be assessed by flow cytometry and H3 incorporation. F) The role of SLE DCs in inducing TH1/TH2 polarization will be evaluated by cytometric bead array. Significance: The results of the studies proposed in this grant might identify potential mechanisms involved in the generation of autoimmune responses and TC abnormalities in SLE. In addition, a better understanding of the molecular basis of DC differentiation and maturation in SLE may provide conceptual tools to regulate DC maturation and/or DC migration to lymphoid organs which may limit excessive stimulation of autoreactive T cells. These could lead into the development of novel therapeutic interventions designed to reverse these abnormalities and abrogate or block the onset and/or severity of this disease.
| Monrad, Seetha U; Rea, Kristine; Thacker, Seth et al. (2008) Myeloid dendritic cells display downregulation of C-type lectin receptors and aberrant lectin uptake in systemic lupus erythematosus. Arthritis Res Ther 10:R114 |
| Monrad, Seetha; Kaplan, Mariana J (2007) Dendritic cells and the immunopathogenesis of systemic lupus erythematosus. Immunol Res 37:135-45 |
| Ding, Dacheng; Mehta, Hemal; McCune, W Joseph et al. (2006) Aberrant phenotype and function of myeloid dendritic cells in systemic lupus erythematosus. J Immunol 177:5878-89 |
