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Macrophages Are the Major Source of Tumor Necrosis Factor in the Porcine Corpus Luteum¹

^a Departments of Obstetrics and Gynecology and ^b Laboratory Medicine, University of South Dakota School of Medicine, Sioux Falls, South Dakota 57105-1570 ^c Center for Reproductive Sciences, Departments of Anatomy and Cell Biology and ^d Molecular and Integrative Physiology, University of Kansas School of Medicine, Kansas City, Kansas 66160

This study was designed to determine the source of tumor necrosis factor (TNF) within the porcine corpus luteum (CL). 1) Sections of frozen or paraffin-embedded CL from various stages of the estrous cycle were incubated with the following primary antibodies: anti-human recombinant TNF, anti-porcine macrophage-specific antigen, or anti--actin (marker of pericyte and smooth muscle cells). Dolichos biflorus lectin-peroxidase was used as an endothelial cell label. Positive immunostaining for TNF was apparent in porcine CL throughout the estrous cycle. TNF immunoreactivity was primarily localized in cells along septal/vascular tracts, and exhibited spatial and temporal distribution similar to that of cells labeled with anti-macrophage antibodies. Large luteal cells exhibited weak staining for TNF in paraffin sections, whereas microvascular endothelial cells were consistently negative in both frozen and paraffin sections. 2) Enriched subpopulations of macrophages, endothelial cells, and large and small luteal cells were isolated by density gradient and immunomagnetic bead separation techniques. TNF secretion by each subpopulation was determined by measuring bioactive TNF in incubation media using a specific in vitro bioassay. Macrophage subpopulations secreted up to 100-fold greater quantities of bioactive TNF (up to 400 pg/10⁶ cells) than did other subpopulations. In contrast, endothelial cell and small luteal cell subpopulations released very small amounts (< 8 pg/10⁶ cells) of bioactive TNF. Large luteal cells secreted slightly greater amounts of TNF (10–15 pg/10⁶ cells). Local macrophages appear to be the primary source of TNF in the porcine CL.

¹ Supported by National Science Foundation grant OSR-9452894 (J.D.B.); NICHD grants CA 50616 (P.F.T.), HD 33994 (P.F.T.), and HD 35333 (J.D.B.); and a USD General Research Fund Minigrant (J.A.B., J.D.B.).

² Correspondence: John Brannian, Dept. of Ob/Gyn, University of South Dakota, Health Sciences Center, 1400 W. 22nd St., Sioux Falls, SD 57105–1570. FAX: 605 357 1528; jbrannia{at}sundance.usd.edu

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