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

Objective: To visualize histochemically the prostaglandin EP1 receptor in human amnion cells and to study the effect of inflammatory cytokines, which are known to stimulate the EP1 receptor, on localization. Methods: Immortalized amnion cells, grown on standard microscope slides and either nonstimulated (control) or stimulated by incubation in culture medium containing interleukin-1β (25 ng/mL), interleukin-4 (50 ng/mL), or tumor necrosis factor alpha (25 ng/mL), were incubated with rabbit anti-human EP1 antibody and stained by a two-step indirect immunoperoxidase strepavidin-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 controls for each reagent, prior blocking with 1% bovine serum albumin or 1% milk, or pretreatment with preimmune rabbit immunoglobulin G were run simultaneously. Slides were viewed by standard light microscopy with and without counterstaining with hematoxylin. Results: Amnion cells incubated in medium alone showed receptor localization throughout the cytoplasmic region of the cell membrane. The localization was nonuniform; a discrete unipolar region of perinuclear nonlocalization was observed. Staining occurred in widely dispersed nests. Cytokine stimulation resulted in increased intensity of staining and an increase in the size of the positive nests; however, it did not affect the discrete unipolar perinuclear region of nonlocalization. Conclusion: Histochemical localization of the human EP1 receptor confirms a cytoplasmic identity and probable plasma membrane localization. Stimulation by inflammatory cytokines increases staining by recruitment of new amnion cells and appears to increase receptor density per cell.