NEUROINTERMEDIATE PITUITARY LOBE CELLS SYNTHESIZE AND RELEASE INTERLEUKIN-6 IN-VITRO - EFFECTS OF LIPOPOLYSACCHARIDE AND INTERLEUKIN-1-BETA

The cytokine interleukin-6 (IL-6) is produced by a variety of cells, i ncluding macrophages, T-cells, and B-cells. Recent studies have confir med a neuroendocrine role for IL-6 in the regulation of anterior pitui tary (AP) hormone release. Because the neurointermediate pituitary lob e (NIL) may modulate AP hormone release, we investigated the productio n of IL-6 by NIL cells in, vitro. NIL tissue removed from pituitary gl ands of male Long-Evans rats was enzymatically and mechanically disper sed, and the cells were subsequently cultured in 96-well tissue cultur e plates for 4-6 days in 10% serum-containing RPMI-1640. Test incubati ons were performed in serum-free RPMI-1640, and IL-6 concentrations we re determined using the 7TD1 cell bioassay. Preliminary studies reveal ed a cell-dependent release of IL-6: increasing the number of NIL cell s per well from 6.25 to 50 x 10(3) revealed detectable basal release o f IL-6 between 25-50 x 10(3) cells/well. The endotoxin lipopolysacchar ide (LPS; 100 ng/ml) and IL-1 beta (100 ng/ml) stimulated IL-6 release at 25 and 50 x 10(3) cells/well. Subsequent studies used a cell densi ty of 50 x 10(3) cells/well and demonstrated time-dependent 3- to 6-fo ld inductions of IL-6 release by 100 ng/ml IL-1 beta and LPS. Concentr ation-response studies revealed maximal stimulation of IL-6 release by 1 ng/ml and a minimally effective concentration of 1 pg/ml for both I L-1 beta and LPS. Treatment of NIL cells with 1-10 mM (Bu)(2)cAMP incr eased IL-6 release by 7- to 14-fold. Endotoxin and IL-1 beta also enha nced the accumulation of IL-6 messenger RNA in these cells. Vasopressi n and oxytocin (1 mu M) inhibited LPS and IL-1 beta stimulation of IL- 6 release from NIL cells, but did not inhibit IL-6 release from AP cel ls. Immunofluorescent dual labeling of NIL cells for flow cytometry re vealed that greater than 95% of the cells did not stain for CD11b/c (c ommon epitope found on monocytes, granulocytes, and macrophages) or CD 45 (leukocyte common antigen). These results demonstrate for the first time the synthesis and release of IL-6 from cultured NIL cells. Agent s that enhance IL-6 release [LPS, IL-1 beta, and (Bu)(2)cAMP] from oth er cell types also increase IL-6 release from NIL cells. Vasopressin a nd oxytocin inhibition of IL-6 release suggests a role for these neuro peptides in feedback inhibition in vivo. Finally, the release of IL-6 is unlikely to be due to leukocytes in the NIL cultures, but is probab ly caused by either pituicytes (modified astroglial cells in the neura l lobe) or melanotrophs of the intermediate lobe.