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, including macrophages, T-cells, and B-cells. Recent studies have confirmed a neuroendocrine role for IL-6 in the regulation of anterior pituitary (AP) hormone release. Because the neurointermediate pituitary lobe (NIL) may modulate AP hormone release, we investigated the production of IL-6 by NIL cells in vitro. NIL tissue removed from pituitary glands of male Long-Evans rats was enzymatically and mechanically dispersed, and the cells were subsequently cultured in 96-well tissue culture plates for 4-6 days in 10% serum-containing RPMI-1640. Test incubations were performed in serum-free RPMI-1640, and IL-6 concentrations were determined using the 7TD1 cell bioassay. Preliminary studies revealed a cell-dependent release of IL-6: increasing the number of NIL cells per well from 6.25 to 50 x 10(3) revealed detectable basal release of IL-6 between 25-50 x 10(3) cells/well. The endotoxin lipopolysaccharide (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 density of 50 x 10(3) cells/well and demonstrated time-dependent 3- to 6-fold inductions of IL-6 release by 100 ng/ml IL-1 beta and LPS. Concentration-response studies revealed maximal stimulation of IL-6 release by 1 ng/ml and a minimally effective concentration of 1 pg/ml for both IL-1 beta and LPS. Treatment of NIL cells with 1-10 mM (Bu)2cAMP increased IL-6 release by 7- to 14-fold. Endotoxin and IL-1 beta also enhanced the accumulation of IL-6 messenger RNA in these cells. Vasopressin and oxytocin (1 microM) inhibited LPS and IL-1 beta stimulation of IL-6 release from NIL cells, but did not inhibit IL-6 release from AP cells. Immunofluorescent dual labeling of NIL cells for flow cytometry revealed that greater than 95% of the cells did not stain for CD11b/c (common epitope found on monocytes, granulocytes, and macrophages) or CD45 (leukocyte common antigen). These results demonstrate for the first time the synthesis and release of IL-6 from cultured NIL cells. Agents that enhance IL-6 release [LPS, IL-1 beta, and (Bu)2cAMP] from other cell types also increase IL-6 release from NIL cells. Vasopressin and oxytocin inhibition of IL-6 release suggests a role for these neuropeptides in feedback inhibition in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)
Spangelo
B L
BL
Department of Physiology, Medical University of South Carolina, Charleston 29425.
deHoll
P D
PD
Kalabay
L
L
Bond
B R
BR
Arnaud
P
P
D000818:Animals; D003994:Bucladesine / Q000494:pharmacology; D002478:Cells, Cultured; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D007375:Interleukin-1 / Q000494:pharmacology*; D015850:Interleukin-6 / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008297:Male; D010121:Oxytocin / Q000494:pharmacology; D010902:Pituitary Gland / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D012485:Salmonella typhi; D014667:Vasopressins / Q000494:pharmacology
D007375:Interleukin-1; D015850:Interleukin-6; D008070:Lipopolysaccharides; D012333:RNA, Messenger; D014667:Vasopressins; D010121:Oxytocin; D003994:Bucladesine