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Effect of protein synthesis inhibitors on leukocytic pyrogen-induced in vitro hypothalamic prostaglandin production.

In order to study the antipyretic effect of inhibitors of protein synthesis, hypothalamic tissue was incubated in vitro under controlled conditions and the amount of prostaglandin E2 (PGE2) measured in the supernatant medium. Rabbit anterior hypothalamic tissue was incubated with purified human leukocytic pyrogen (LP) and after 60 minutes the supernatant fluid was assayed for PGE2 by radioimmunoassay. Control tissue incubated with Eagle's medium (MEM) released elevated levels of PGE2; however, the addition of polymyxin B (PmxB), a cationic antibiotic which blocks the activities of bacterial endotoxins, significantly reduced PGE2. In addition, endotoxin added to MEM induced from the brain tissue PGE2 production which could be reduced by the addition of PmxB. Thus, commercial culture media such as MEM may contain sufficient amounts of endotoxin to stimulate brain PGE2 production in vitro. Purified human LP incubated with hypothalamic tissue in the presence of PmxB induced PGE2 production in a dose-dependent fashion. This release could be reduced (p less than 0.001) by the presence of either cycloheximide or puromycin during incubation with LP. The addition of these inhibitors to unstimulated hypothalamic tissue incubations did not reduce background levels of PGE2. It is concluded that the antipyretic effect of protein synthesis inhibitors results in a specific decrease in LP-induced levels of PGE2.


INTRODUCTION
Substantial evidence exists that leukocytic pyrogen (LP) produces fever by first stimulating the production of prostaglandin E (PGE) in the anterior hypothalamus [ 1,2,3]. Once released, PGE and possibly other cyclo-oxygenase products bring about a rise in the temperature set-point of the hypothalamus with the subsequent development of a fever by physiologic (e.g, shivering and vasoconstriction) and behavioral means [4]. Recently we have shown that purified LP induces the synthesis of PGE2 from rabbit brain tissue in vitro [5]. This response is dose-related and can be eliminated by inactivation of LP with moderate heating. An advantage of this in vitro assay is that the effects of LP-induced PGE2 synthesis as well as other neurochemical changes can be directly studied under controlled conditions.
In addition to inhibitors of cyclo-oxygenase such as aspirin and indomethacin, inhibitors of protein synthesis such as cycloheximide (Cyhx) and anisomycin have been shown to act as antipyretic agents [6]. Injection of Cyhx into rabbits prevents fever due to intravenous injection of endotoxin or LP [7,8,9]. The mechanism of this antipyresis, however, is uncertain. With this fact in mind, the current study was undertaken to examine the effects of protein synthesis inhibitors (e.g., puromycin and Cyhx) on the LP-induced production of PGE2 from rabbit hypothalami. The results indicate that Cyhx and puromycin decrease LP-induced hypothalamic PGE2 release. This response may be due to the ability of protein synthesis inhibitors to decrease the synthesis of enzyme(s) needed for prostaglandin synthesis.

METHODS AND MATERIALS Purification ofHuman LP
Human LP was produced from plateletpheresis by-products rich in monocytes. The mononuclear cells were separated, suspended, and stimulated with heat-killed Staphylococcus albus as previously described [10]. The crude 24-hour supernatant was first fractionated using a Millipore Pellicon concentrating device (Millipore, Bedford, MA) which excluded molecules greater than 100 kD and concentrated molecules between 100 and 10 kD. This concentrate was then dialyzed against pH 7.4 phosphate-buffered saline and passed over an anti-human LP affinity column; the LP was eluted with citric acid as previously reported [11]. This material was then concentrated in 10 percent lysine HCI and chromatographed over Sephadex G-50 (fine), 170 x 5.0 cm in 0.15 M NaCl at 40C. The 15 kD peak of pyrogen activity was isolated and concentrated in 3.5 kD cut-off dialysis bags submerged in polyethylene glycol, 8-10 kD. An aliquot of this material, when precipitated with trichloracetic acid and applied to 17 percent polyacrylamide gels in SDS, revealed a single band stained with Coomassie blue. LP intended for biological assay was not run on gels but assayed in New Zealand albino rabbits by intravenous injection. Rectal temperatures were recorded every minute using indwelling Yellow Spring thermistors (Yellow Spring Instruments, Yellow Spring, OH) on a Digistrip II constant recording device (Kaye Instruments, Bedford, MA). Each lot of purified LP was assayed in 2 kg rabbits, and peak fevers between 0.6 and 0.90C were considered a rabbit pyrogen dose (RPD). Using this method, peak fever in 100 rabbits occurred at 46.9 ± 8.9 minutes (SD). Each lot of LP was aliquoted and kept frozen at -220C until used.

Production ofPGE2 by Rabbit Brain Tissue
For each experiment, a rabbit was sacrificed by rapid CO2 asphyxiation. Within one minute, the spinal cord was severed and the brain was removed from the rostral to frontal end, severing the pituitary stalk. The anterior hypothalamus was excised and quickly placed in ice-cold Eagle's minimal essential medium (MEM, Microbiological Associates, Walkerville, MD, containing 0.01 M HEPES buffer, 100 units penicillin G/ml, and 100 ,ug streptomycin/ml) as previously described [5]. The optic chiasm was dissected away and discarded. The hypothalamic tissue was then weighed, minced into 0.5 mm3 sections, and suspended in MEM at a concentration of 10 mg/ml. 1.0 ml aliquots of the suspension were then added to 12 x 75 mm polypropylene tubes (Falcon Plastics, Oxnard, CA). The aliquots were incubated for 30 minutes in a 370C shaking water bath. After this time, 3 ml of warm MEM were added to each tube and the suspensions then centrifuged at low speed (5 g) for five minutes. The supernatant fluid was discarded and 1 ml of fresh MEM, containing appropriate dilutions of various materials (that is, LP, endotoxin, or protein synthesis inhibitors), was added to duplicate tubes. The suspensions were then reincubated in the shaking water bath for an additional 60 minutes. Supernatants from these incubations were obtained by low-speed centrifugation and immediately frozen in dry ice and stored at -700C until assayed. Supernatants from each tube were assayed in duplicate for PGE2 with a radioimmunoassay kit containing a specific antibody for PGE2 (Seragen, Boston, MA), thus yielding four data points for each experimental incubation condition. The means of the duplicate PGE2 assay results were used to calculate the mean for each duplicate incubation. Results are expressed as pg of PGE2/mg of tissue ± SEM. The data analysis utilized the paired student's t-test.

Reagents
Cycloheximide was purchased from Boehringer-Mannheim and dissolved in MEM at a stock solution of 500 ,ug/ml and kept at -700C. This was diluted in MEM to final concentrations and added to brain minces. Puromycin was obtained from Sigma and dissolved in MEM as described above for Cyhx. Polymyxin B was purchased from Pfizer, dissolved in pyrogen-free water, and stored at 1 mg/ml at 40C. Endotoxin (E. coli RE-2) was obtained from the Bureau of Biologics (Bethesda, MD). For endotoxin levels, the limulus amebocyte lysate test was used (Associates of Cape Cod, Woods Hole, MA).

RESULTS The Effect ofPolymyxin B on Hypothalamic PGE2 Production Induced by
Endotoxin or LP Endotoxin induces the release of LP both in vitro and in vivo. In additon, there are several experiments which suggest that endotoxin can also act directly on the brain and induce fever; recently endotoxin has been shown to stimulate brain cells to synthesize PGE2 in vitro [5]. Since endotoxins are seemingly ubiquitous contaminants of many laboratory reagents, it was not surprising to detect significant levels of endotoxin in the MEM used in these studies. Endotoxin levels from 0.1-1 ng/ml were measured in six different lots of MEM. Polymyxin B, a positively charged antibiotic, has been shown to block or inhibit many of the multiple biological activities of endotoxins, including the induction of LP in vitro and in vivo [12,13]. Sections of hypothalami were incubated with either MEM alone, MEM containing 15 ,ug/ml of PmxB, MEM to which endotoxin had been added (30 pg/ml), or MEM containing both endotoxin and PmxB. As shown in Fig. 1, background levels of PGE2 were elevated in control incubations of hypothalamic tissue in MEM. However, PmxB added to the MEM resulted in significantly lower levels of PGE2. As an additional test, known amounts of endotoxin were added to MEM both in the absence and the presence of PmxB. As shown in Fig. 1, PmxB inhibits this endotoxin-induced release of PGE2 from rabbit brain tissue.
The next experiments involved the dose-response effects of human LP in inducing PGE2 from rabbit hypothalami in the presence of these same concentrations of PmxB. As shown in Fig. 2, PmxB had no effect on LP-induced PGE2 production. In these experiments, four different lots of human LP purified by the same methods were used. In each case the activity of the LP in producing fever in the rabbit following intravenous injections was compared to the induction of PGE2 in vitro. As shown, I0O RPD induced significant PGE2 production (p < 0.05). Effects  of each other at room temperature. These incubations were then returned to the water bath for 60 minutes as described in Methods. The results as depicted in Fig. 3 show that Cyhx does not inhibit the background release of PGE2 from brain tissue (p > 0.2).
On the other hand, Cyhx significantly reduces the release of PGE2 from hypothalamic tissue incubated with purified human LP (p < 0.001). The ability of Cyhx to reduce LP-induced PGE2 production was consistently observed in all experiments using all lots of LP.
In order to further clarify whether Cyhx was having a toxic effect on the brain cells or interfering with cell metabolism, an additional series of experiments was carried out in which the effect of Cyhx on PGE2 production during the preincubation was studied. Cyhx was added to brain tissue either both during and after the preincubation step or only after the preincubation. Regardless of when Cyhx was added to the tissue, background levels of PGE2 were not significantly reduced (Fig. 4,  Cyhx reduced LP-induced release of PGE2 by similar amounts regardless of whether the protein synthesis inhibitor was added at the same time as the LP or was present before and after the addition of LP (Fig. 5).
Cycloheximide has been described as toxic in that when given systemically it can interfere with the ability of rats and rabbits to maintain normal body temperature [14, 9,15]. Although our experimental design does not reflect possible peripheral effects of Cyhx, we used another inhibitor of protein synthesis, puromycin. Incubation of hypothalamic tissue with this drug (2 or 10 ,ug/ml) did not significantly affect the background release of PGE2 (p > 0.2, Fig. 6). These concentrations of puromycin, like Cyhx, are consistent with the concentrations used in several cell culture systems to block protein synthesis in a non-toxic, reversible system [16]. Similar  obtained using Cyhx, however, the additon of puromycin to brain tissue incubated with LP resulted in a 50 percent reduction in the amount of PGE2 detected in the supernatant after 60 minutes (Fig. 6). DISCUSSION The initial results of these studies indicate that commercial media such as MEM probably contain sufficient levels of endotoxin to affect our experimental conditions by stimulating brain PGE2 production in vitro. The addition of polymyxin B to the media eliminates or significantly reduces the ability of such contaminating endotoxins to induce PGE2 release or production from rabbit hypothalamic tissue. The antibiotic, however, does not affect the ability of LP to induce the synthesis of PGE2 from rabbit hypothalamic tissue, and these results are consistent with previous studies demonstrating that LP and endotoxin effects on in vitro PGE2 production are separable [5]. Not all lots of MEM contain the same amounts of endotoxin, and thus background levels of PGE2 induced by media alone may vary from experiment to experiment. This problem was overcome by including polymyxin B in the culture media of all subsequent incubations and provided the opportunity to carry out these studies without endotoxin contamination as a contributing factor. As shown by other investigators [12,13], the addition of this antibiotic to culture media is especially important when various agents are employed in pyrogen experiments for their ability to stimulate the production and release of LP. We can now add that production of PGE2 from brain, whether in vitro or in vivo, can also be affected by contaminating endotoxins and that the inclusion of polymyxin B can block potential endotoxin effects.
Once we were able to obtain consistent results using PmxB in the hypothalamic incubations, studies were carried out in order to investigate the effect of the antipyretic protein synthesis inhibitors on brain PGE2 production in vitro. Cycloheximide, a potent inhibitor of protein synthesis in vitro, has been used as a chemotherapeutic agent in certain cancers, and patients receiving the drug during a febrile illness undergo a dramatic reduction in fever within an hour following an infusion of the drug [6]. In addition, administration of Cyhx to animals receiving either endotoxin or LP eliminates or lowers their fevers [7,8,9]. Investigations have shown that this antipyresis is not due to Cyhx preventing pyrogens from entering the brain from the circulation since fevers produced by LP or endotoxin given intracerebroventricularly (ICV) can still be inhibited by Cyhx [17,3]. Moreover, the doses of Cyhx which are antipyretic in rabbits given exogenous pyrogens are not sufficient to prevent the synthesis of LP in vivo [8]; however, this is in contrast to the fact that both Cyhx and puromycin block LP synthesis when added to phagocytes at the time of stimulation with exogenous pyrogens in vitro [ 1 6,18]. The mechanism for protein synthesis inhibitors acting as antipyretics remains unclear. Siegert et al. [8] have shown that Cyhx reduces fever without affecting cerebrospinal fluid levels of PGE2. However, recent results have shown that Cyhx does not interfere with the production of fever caused by the administration of PGE2 or arachidonic acid [17,19]. In addition, Cyhx inhibits the in vitro release of prostaglandins from mouse BALB/3T3 cells treated with methylcholanthrene [20], while various other protein synthesis inhibitors reduce the LP-induced release of PGE2 from human dermal fibroblasts [21,22]. These later findings as well as those reported in this paper indicate that protein synthesis inhibitors may exert their antipyretic effect by interfering with the synthesis of PGE2.
In the present studies, we consistently observed that hypothalamic tissue incubated with Cyhx and LP released significantly less amounts of PGE2 than tissue incubated with LP alone. In addition, background levels of PGE2 released by these tissues were unaffected by the presence of Cyhx. Similar results were obtained when another protein inhibitor, puromycin, was used. These data do not rule out the possibility that protein synthesis inhibitors are toxic to brain tissue and thereby decrease its ability to respond to LP; on the other hand, recent evidence shows that protein synthesis inhibitors are non-toxic to human dermal fibroblasts [21]. In these experiments, the ability of fibroblasts to release PGE2 in response to LP was decreased by the presence of Cyhx; however, fibroblasts incubated for various time periods with Cyhx retained their viability and showed no decrease in background PGE2 production. These results support the concept that inhibitors of protein synthesis block some synthetic step leading to LP-induced release of PGE2 rather than simply injuring or killing the responding cells.
The synthetic step blocked by protein synthesis inhibitors is unknown. However, since fibroblasts synthesize PGE2 in the presence of both arachidonic acid and protein synthesis inhibitors it is unlikely that inhibitors of protein synthesis interfere signifi-cantly with the activity of cyclo-oxygenase (preliminary data and [211). On the other hand, it may be that LP induces PGE2 release by first activating a phosphodiesterase enzyme which cleaves diacylglycerol (DAG) from phosphotidylinositol [231. The DAG so formed may have arachidonic acid on its 2-position, which in turn could be cleaved by a specific enzyme (a diacylglycerol lipase and/or a phosphatidic acid specific phospholipase-23). Protein synthesis inhibitors may therefore decrease the LP-induced release of PGE2 by interfering with the synthesis of one or more of the enzymes involved in the release of arachidonic acid.
Another protein synthesis inhibitor, anisomycin, which is less toxic than Cyhx in that it does not interfere with normal thermoregulation of rats or rabbits [24], blocks or significantly reduces fever induced by either endotoxin or LP [25,26]. Unlike Cyhx, however, anisomycin also prevents fevers induced by PGE2 [27]. In addition, after administration of endotoxin, anisomycin treatment results in an increased amount of PGE2 in the cerebrospinal fluid [3]. It is unclear whether the same results would be obtained if fever were induced by an injection of LP instead of large intravenous doses of endotoxin which probably induce PGE2 production from cells throughout the brain [5]. Recent results from this laboratory [22] and elsewhere [21] indicate that anisomycin inhibits the LP-induced release of PGE2 from fibroblasts and may, therefore, also decrease the LP-induced release of PGE2 from hypothalamic tissue. It is also possible that inhibitors of protein synthesis which are also antipyretics may not induce antipyresis by the same mechanism.