Immunohistochemical localization of the prostaglandin E subtype-1 receptorin cytokine-stimulated and unstimulated amnion cells

Objective: To visualize histochemically the prostaglandin EP1 receptor in h uman amnion cells and to study the effect of inflammatory cytokines, which are known to stimulate the EPI receptor, on localization. Methods: Immortalized amnion cells, grown on standard microscope slides and either nonstimulated (control) or stimulated by incubation in culture medi um containing interleukin-1 beta (25 ng/mL), interleukin-4 (50 ng/mL), or t umor necrosis factor alpha (25 ng/mL), were incubated with rabbit anti-huma n EP1 antibody and stained by a two-step indirect immunoperoxidase strepavi din-biotin method using horseradish peroxidase and 3,3' diaminobenzidine as the chromogen. The localization was done on ten different flasks of cells. Duplicate slides for each cytokine concentration were prepared. Negative c ontrols for each reagent, prior blocking with 1% bovine serum albumin or 1% milk, or pretreatment with preimmune rabbit immunoglobulin G were run simu ltaneously. Slides were viewed by standard light microscopy with and withou t counterstaining with hematoxylin. Results: Amnion cells incubated in medium alone showed receptor localizatio n throughout the cytoplasmic region of the cell membrane. The localization was nonuniform; a discrete unipolar region of perinuclear nonlocalization w as observed. Staining occurred in widely dispersed nests. Cytokine stimulat ion resulted in increased intensity of staining and an increase in the size of the positive nests; however, it did not affect the discrete unipolar pe rinuclear region of nonlocalization. Conclusion: Histochemical localization of the human EP1 receptor confirms a cytoplasmic identity and probable plasma membrane localization. Stimulatio n by inflammatory cytokines increases staining by recruitment of new amnion cells and appears to increase receptor density per cell. (Obstet Gynecol 1 999;94:1027-32. (C) 1999 by The American College of Obstetricians and Gynec ologists.).