The production of collagenase (EC 3.4.24.3) by endotoxin-stimulated macrophages was significantly inhibited by indomethacin, indicating that prostaglandins (PGs) mediate this effect. Inhibition of collagenase production by indomethacin was overcome by addition of exogenous PGE2 at 10 nM whereas the addition of 0.1 and 1.0MgM PGE2 increased the enzyme production to 3 times that achieved by endotoxin. Although the addition of prostaglandin alone to macrophage cultures did not stimulate collagenase production, the simultaneous addition of PGE1 or PGE2 and endotoxin enhanced collagenase activity 2-to 10-fold. This increase was detectable at PGE concentrations of 10 nM and was optimal at 0.1-1.0 ,uM.PGF2, had little effect on either the enhancement of collagenase production by endotoxin-stimulated macrophages or its restoration in cultures inhibited by indomethacin. Radioimmunoassay of prostaglandins in the culture media revealed that macrophages exposed to endotoxin secreted 40-fold more PGE2 than did unstimulated cells. The increase in PGE2 was detected 4 hr after exposure to endotoxin and was maximal at 14 hr. The peak PGE2 concentrations in the culture media were similar to those of exogenous PGE2 (about 10 nM) needed to restore collagenase production in indomethacin-treated cultures. These findings demonstrate the involvement of PGE in the endotoxin-activation of macrophages with resultant production of collagenase.Certain chronic inflammatory lesions are often accompanied by destruction of connective tissue. Increased levels of collagenase (1-6) and prostaglandins (779) have been identified in many of these lesions. The coexistence of these two components in sites of inflammation suggests that prostaglandins may influence collagenase production. Since we have previously shown that the macrophage, a prominent cell type in chronic inflammatory lesions, can be stimulated by endotoxin (10) or lymphokines (11) to produce collagenase, it was of interest to determine whether prostaglandins were involved in the activation of these cells. Here we report that the elevated levels of prostaglandin E2 (PGE2) produced by endotoxin-stimulated macrophages have a regulatory role in the production of collagenase by these cells. MATERIALS AND METHODSCulture Methods. Macrophages were obtained from mineral oil-induced peritoneal exudates in guinea pig and cultured as described (10). Activation of the macrophages was achieved by the addition of endotoxin (30 ,ug/ml) from Escherichia coli (055:B5 Difco Laboratories, Detroit, MI) to the cultures. Media were harvested daily from the cultures and frozen at -20°until assayed. Prostaglandins E1, E2, and F2a (PGE1, PGE2, and PGF2a) (generously supplied by John Pike, Upjohn Co., Kalamazoo, MI) were dissolved in ethanol and added to the culture The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. (12). One volume of 1 M sodium citrate (pH 3.5) was adde...

MATERIALS AND METHODS Cell CultureGuinea pig macrophages were obtained from peritoneal exudates induced by intraperitoneal injection of sterile mineral oil (Drakeol, 6VR, Penreco, Inc., Butler, PA) into male Hartley guinea pigs.' The macrophages were purified by adherence and cultured as previously described.z Activation of the macrophages in culture was achieved by exposure of the cells to endotoxin (lipopolysaccharide) from Escherichia coli 055:B5 (Difco Lab., Detroit, MI). Some of the cultures received indomethacin (Merck, Sharp and Dohme Research Lab., Rahway, NJ) , isobutyl-methyl-xanthine (IBMX) or epinephrine (Sigma, St.Louis, MO) . Annals New York Academy of Sciences Prostaglandin AssayPG levels in the media were determined by direct radioimmunoassay utilizing anti-PGE, serum (Miles, Elkhart, IN). PGE, was a gift from Dr. John Pike (Upjohn Company, Kalamazoo, MI). Cyclic A M P AssayCyclic AMP was determined utilizing 2 X lo7 macrophages plated in 25 cm2 flasks. Following removal of the media from the cultures, the cAMP from the adherent cells was extracted by the addition of 3 ml of 2% HCIO,. The extracts were boiled, centrifuged at 400 X g for 5 minutes and the supernatants neutralized with 3M Tris. These supernatants were chromatographed on neutral alumina (Fisher Scientific, Silver Spring, MD) and Bio-Rad AG-1X-2 (200-400 mesh, chloride form) (Bio-Rad Laboratory, Rockville Center, N.Y.), columns.1o The cAMP eluted with 0.05 N HCI was lyophilized and assayed by a modified radioirnmunoassay,ll utilizing anti-CAMP serum (Collaborative Research, Waltham, MA). Collagenase ActivityCollagenase activity in the culture media was assayed utilizing [14C]glycinelabeled collagen fibrils as described.* RESULTSAs shown in TABLE 1, macrophages stimulated with endotoxin produce collagenase. The levels of collagenase were significantly enhanced by the

Chromatographic and functional properties of tyrosine isoaccepting transfer ribonucleic acids (tRNAs) from placenta and HeLa cells were analyzed and compared. RPC-5 chromatography separated four major isoacceptors from each source, with those from HeLa cells eluting generally later than those from placenta. There was some overlap: HeLa tRNA1Tyr eluted in a position between placenta tRNA3Tyr and tRNA4Tyr; and HeLa tRNA2Tyr and placenta tRNA4Tyr eluted in similar positions with the HeLa isoacceptor eluting slightly later than the placental isoacceptor. Thus there are no isoacceptors common to both sources. The function of the individual isoacceptors was compared in a rabbit-reticulocyte, cell-free, protein-synthesizing system for both the rate of incorporation of tyrosine into the polypeptide chain and the site of incorporation in alpha-globin. Two isoacceptors were compared simultaneously in the same reaction, and overlapping comparisons were made to relate each isoacceptor to all the others. There were no significant differences in the rates of incorporation among the isoacceptors, nor were there any differences in the sites of incorporation. All eight isoacceptors donated tyrosine equally well into the three tyrosine containing tryptic peptides of alpha-globin. Whatever the structural differences among the tyrosine isoacceptors are, they do not affect the function of the tRNA in this protein-synthesizing system.