Date of Award

2009-01-01

Degree Name

Master of Science

Department

Biological Sciences

Advisor(s)

KRISTINE M. GARZA

Abstract

Leptin is a pleiotropic hormone primarily secreted by white adipose tissue. Although originally found to regulate food intake and energy expenditure, it is now known to also significantly affect the immune system. In this study we assessed the effect of leptin on bone marrow derived dendritic cell (BM-DC) cytoskeletal structure and functionality. Our preliminary data demonstrates that the addition of physiological concentrations of exogenous leptin to BM-DC causes cytoskeletal rearrangement specifically that of actin as evidenced by increased number of llamelopodia/dendrites and increased staining for actin within the llamelopodia/dendrite region. This suggests that leptin is a necessary component of DC - T cell interaction in that leptin may be responsible for enhanced migration of the DC to draining lymph nodes and potentiating the extent of the physical contact between these cells. We therefore hypothesized that leptin increases the number and length of dendrites in DCs by causing a re-arrangement of the cytoskeleton, specifically actin, which in turn leads to an increased capacity to migrate and to activate T cells. To test this hypoThesis we used BM-DCs from C57Bl/6 (B6) mice enriched by magnetic bead cell sorting. The effect of leptin on BM-DC cytoskeletal re-arrangement was assessed by light and confocal microscopy; actin polymerization was determined by Western blot analysis for cell content of F-actin versus G-actin in fractioned cellular lysates of treated cells. The functional effects of leptin on BM-DC were evaluated in-vitro by assessing DC migration and DC ability to active antigen-specific T cells. The differentially treated BM-DC were evaluated in transwell migration assays to assess their ability to migrate towards a DC-specific chemokine CCL19. Finally, treated BM-DC ability to activate T cells was assessed in-vitro by measuring activation of an antigen-specific T cell hybridoma or of antigen-specific primary T cells (both specific to ovalbumin) as a function of IL-2 and INF-gamma production or proliferation, respectively. We found that there is only a slight increase in actin content within the cell post treatment of leptin, however this increase is not significant. The addition of an exogenous physiological concentration of leptin to BM-DC enhanced polymerization of actin and therefore the formation of dendrites, both in number and length and thickness. This effect was particularly evident in the leptin and LPS duo treatment. Functionally the presence of leptin appears to enhance migration and promote T cell activation. This is likely due to the increased surface area, as well as dendrite number length and thickness which increase the likelihood of DC-T cell interactions, therefore potentiating the extent of the physical contact between these cells. This is further supported by enhanced T cell activation by leptin and LPS treated cells as measured by incorporation of tritiated thymidine, IL-2 and INF-gamma production. The data obtained from this study suggest leptin plays an important role in immunological responses, and may specifically contribute to the pro-inflammatory state and increased susceptibility of autoimmune disorders found in obesity.

Language

en

Provenance

Received from ProQuest

File Size

99 pages

File Format

application/pdf

Rights Holder

CHRISTINE DELGADO

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