Effect of Alpha 2 Macroglobulin on Luteal Cell Induced Gamma Delta T Lymphocyte Proliferation.

Abstract

The paracrine factors that regulate communication between immune cells and parenchymal cells within the corpus luteum (CL) are not well defined. It has been shown that cultured luteal cells are profound stimulators of T lymphocytes and can induce T cell proliferation by cell to cell interactions and by secretory factors. By using a proteomic approach, we profiled protein expression in conditioned media from gamma delta T cell - luteal cell (LC) cocultures collected from midcycle and regressing CL. Alpha 2 macroglobulin (a2M) was one of the proteins that exhibited differential secretion. The purpose of this study was to determine if a2M mediates LC induced changes in gamma delta T cell function in fully functional, but not regressing CL. The first objective was to define if a2M secretion can be modified by luteolytic factors. Midcycle luteal cells were exposed to transforming growth factor beta(TGFb), interferon gamma/ tumor necrosis factor (IFNg/TNFa) and PGF2a. Luteal conditioned media (LCM) were collected on days 1 and 3 of cultures and protein expression was analyzed by Western Blot. Although PGF2a reduced a2M secretion in both days of collection, the effect of IFNg/TNFa was observed only on day 3 (p<0.05). The second objective was to determine if a2M mediates gamma delta T cells proliferation in response to luteal cells depending on functional status of the CL. Corpora lutea were collected from cyclic cattle on days 10–12 of the estrous cycle and 8hrs after PGF2a injection, dissociated and placed in culture. To test two different ways of cells communication, immunomagnetically isolated gamma delta T cells from the blood were placed in culture with luteal cells in contact or inside a culture insert. Cells were than incubated for 72hrs with or without a2M and proliferation of gamma delta T cells was measured. In cells collected from regressing CL, a2M increased LC stimulated gamma delta T cells proliferation only when the cells were physically separated by insert. The gamma delta T cells response to midcycle LC was not altered by a2M. The third objective was to examine if a2M can induce gamma delta T cells proliferation directly. The luteal cells were first fixed with 0.1% and 1% paraformaldehyde. Gamma delta T cells and a2M were then added to the culture system and a proliferation assay was performed after 72 hrs. This study showed that cell fixation reduced gamma delta T cells response to LC respectively to 40% and 23%. Treatment with a2M had no effect on gamma delta T cell proliferation. In conclusion, we showed that a2M protein expression varied within the estrous cycle and was regulated by factors locally secreted in the CL. The role of a2M is to entrap and bind diverse molecules secreted by luteal cells and facilitate their delivery to immune cells. Our data suggests that proteins like a2M may provide an alternative mechanism for cell communication in tissues undergoing rapid morphological changes to maintain local homeostasis. This project was supported by National Research Initiative Competitive Grant no. 2008-35203-04617 from the USDA National Institute of Food and Agriculture.