Coincidental Changes in Behavior and Plasma Cortisol in ...

0013-7227/97/$03.00/0 Endocrinology Copyright © 1997 by The Endocrine Society

Vol. 138, No. 6 Printed in U.S.A.

Coincidental Changes in Behavior and Plasma Cortisol in Unrestrained Pigs after Intracerebroventricular Injection of Tumor Necrosis Factor-a* E. J. WARREN, B. N. FINCK, S. ARKINS, K. W. KELLEY, R. W. SCAMURRA, M. P. MURTAUGH, AND R. W. JOHNSON Laboratory of Integrative Biology (E.J.W., B.N.F., R.W.J.) and Laboratory of Immunophysiology (K.W.K.), Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801; Department of Biological Sciences (S.A.), Illinois State University, Normal, Illinois 61790; and Department of Veterinary PathoBiology (R.W.S., M.P.M.), University of Minnesota, St. Paul, Minnesota 55108 ABSTRACT The coincidental behavioral and physiological responses to inflammatory stimuli administered either peripherally or centrally were evaluated. In the first study, twenty castrated male pigs were injected ip with 0, 0.5, 5, or 50 mg/kg BW lipopolysaccharide (LPS). Body temperature was monitored telemetrically, and serial blood samples were collected via an indwelling jugular catheter for determination of plasma cortisol and tumor necrosis factor-a (TNF-a) concentrations. Sickness behaviors were measured during 10-min tests at 0, 2, 4, 8, 12, and 24 h post injection. The 5 and 50 mg/kg doses of LPS increased plasma concentrations of cortisol and TNF-a, while inducing anorexia, hypersomnia, and fever. In contrast, although 0.5 mg/kg LPS induced acute anorexia, hypersomnia, and fever, it did not increase plasma TNF-a; and the cortisol response was small and transient, suggesting the behavioral system in pigs is more responsive to LPS

than the hypothalamic-pituitary-adrenal (HPA) axis. Because LPSinduced behavior and activation of the HPA axis involve proinflammatory cytokines in the brain, in a second study, unrestrained pigs with jugular catheters were injected intracerebroventricularly (ICV) with recombinant porcine TNF-a. Vehicle or TNF-a (0, 5, or 50 ng/kg) was injected ICV, and plasma cortisol and behavior were determined as before. Pigs injected ICV with 50 ng/kg TNF-a showed anorexia, hypersomnia, and an abrupt increase in plasma cortisol concentration. Whereas 5 ng/kg TNF-a ICV also induced marked sickness behavior, it failed to stimulate the HPA axis, as indicated by plasma cortisol levels. That there was a distinct difference in the magnitude of behavioral and endocrine responses to LPS and TNF-a suggests that different systems that are responsive to inflammatory stimuli exhibit different sensitivities. (Endocrinology 138: 2365–2371, 1997)

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acute infection is now confirmed by numerous independent studies in animals involving peripheral administration of recombinant cytokines (4, 5). Whether TNF-a and other cytokines in peripheral blood directly access the CNS has been the subject of considerable debate (6). Regardless, it is clear that TNF-a and its receptors are present in the CNS. For instance, increased TNF-a synthesis in brain has been observed in the animal model for multiple sclerosis (i.e. experimental allergic encephalomyelitis) (7), head injury or trauma (8), and after peripheral injection of lipopolysaccharide (LPS) (9, 10). The cytokine is likely produced by activated microglial cells that are present in the CNS and, like macrophages, are derived from mononuclear myeloid progenitors (11). Within the CNS, TNF-a induces fever through a direct action on hypothalamic neurons and by triggering the release of IL-1 (12). It also induces anorexia (13) and is involved in the release of ACTH by the pituitary (14, 15). Binding sites for TNF-a have been identified in the brainstem, cortex, cerebellum, thalamus, and basal ganglia of the rat brain (16). Thus, there is considerable evidence indicating that TNF-a contributes to the behavioral and neuroendocrine changes that characterize sickness. Most of the studies concerning the behavioral and neuroendocrine effects of proinflammatory cytokines have been conducted in rodents. However, experimental limitations of rodent models and the growing interest in a clinically rele-

LINICAL symptoms of sickness (such as anorexia, fever and lethargy) have been repeatedly observed in animals with autoimmune, infectious, or neoplastic disease. These seemingly heterogenous diseases have in common, conditions that are likely to result in the release of major proinflammatory cytokines, including tumor necrosis factor-a (TNF-a). Synthesized by mononuclear myeloid cells as a 26-kDa protein, membrane-bound TNF-a undergoes processing by a metalloproteinase to produce the secreted, biologically active 17-kDa form (1). The first suggestion that TNF-a and other products from activated leukocytes affect the central nervous system (CNS) and alter behavior came from clinical trials, where recombinant cytokines were injected peripherally to potentiate immune responses or treat neoplastic disease (2, 3). These efforts were terminated when patients receiving TNF-a and interferon-a developed severe flu-like symptoms, including anorexia, fever, malaise, and lethargy. The involvement of an immunogenic component, rather than a pathogenic component, in the central effects of Received November 5, 1996. Address all correspondence and requests for reprints to: Dr. Rodney W. Johnson, Laboratory of Integrative Biology, Department of Animal Sciences, University of Illinois, 1207 West Gregory Drive, Urbana, Illinois 61801. E-mail:[email protected]. * This research was supported in part by grants from the NIH (DK 51576), National Pork Producer Council (1341), Illinois Pork Producers Association, and USDA, CSRS (Hatch 35–319; AH&D 35–969).

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COINCIDENTAL RESPONSES TO LPS AND TNF-a

vant outbred animal model have prompted the use of the pig as an alternative for many immunophysiological investigations. Swine, which are similar to humans in size, physiology, and dietary habits, are now used for studying, among other things: 1) the suppressive effects of opiate drugs on humoral and cell-mediated immune responses (17); 2) adult respiratory distress syndrome in sepsis (18); and 3) organ transplantation between discordant species (19). The pig also affords opportunities for studying the role of cytokines in the pathogenesis of CNS infections and autoimmune diseases. For example, Nam et al. (20) used cultured porcine astroglia to demonstrate the rapid induction of PG F2a production in response to recombinant human IL-1a, whereas Megyeri et al. (21) used a neonatal piglet model to show that intracisternal injection of recombinant human TNF-a increased the permeability of the blood-brain barrier to sodium-fluorescein. Although porcine macrophages produce the characteristic array of proinflammatory cytokines in response to antigen in vitro (22, 23), neither the secretion of TNF-a in response to in vivo challenge nor the behavioral and physiological effects of TNF-a in the pig brain have been adequately explored. In the present study, we sought to address these issues and, in doing so, take advantage of the pig model to simultaneously and chronologically evaluate behavioral and physiological responses to inflammatory stimuli administered either peripherally or centrally. We recently demonstrated in the rat that the high sensitivity of the hypothalamic-pituitary-adrenal (HPA) axis to LPS, and the resultant increase in plasma corticosterone, are important mechanisms for preventing profound behavioral disturbances in response to low-grade immune stimulation (24, 25). In contrast, in the present study, by monitoring the coincidental changes in behavior and plasma cortisol after peripheral injection of LPS or central injection of TNF-a, we found evidence that suggests that the HPA axis in pigs is not as responsive to inflammatory stimuli. Because glucocorticoids inhibit LPS-induced sickness behavior (24 –26), the present data suggest that the relative insensitivity of the pig’s HPA axis to LPS and TNF-a contributed to the rather profound anorexia and hypersomnia that were observed. Materials and Methods Experimental animals Castrated male crossbred pigs, weighing 15–25 kg, were housed individually in stainless-steel cages (0.75 m 3 0.75 m). They were maintained at 24 C under a 16-h light, 8-h dark cycle (lights on at 0600 h). Water and a pelleted corn and soybean meal-based diet were available ad libitum unless otherwise stated. All care met or exceeded requirements in the Guide for Care and Use of Agricultural Animals in Teaching and Research (27).

Surgical procedures Anesthesia was induced with sodium thiamylal (20 mg/kg BW iv) and maintained with halothane (2–5%) and oxygen (300 cc/min). Pigs were administered penicillin (600,000 units im) postsurgically and were allowed at least 7 days of recovery before beginning an experiment. All procedures were approved by the University of Illinois Laboratory Animal Care Advisory Committee. Jugular catheterization. The right jugular vein was surgically exposed and a catheter (Tygon microbore tubing; id, 1.02 mm; od, 1.78 mm; Fisher

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Scientific, Pittsburgh, PA) was advanced posteriorly 15–20 cm to the vena cava and sutured in place. The distal end of the catheter was directed sc to the dorsal neck region and exteriorized between the scapulae for repeat blood sample collection. Catheters were flushed daily with 5 ml sterile heparinized saline (4 U/ml) to prevent occlusion. Intracerebroventricular (ICV) cannulation. Pigs requiring a central injection had a cannula stereotaxically placed into the left lateral cerebral ventricle (ICV), as previously described for swine (28). Briefly, the head of the pig was oriented in a large-animal stereotaxic instrument so that the plane formed by the frontal and parietal bones was parallel to the instrument table top. An 18-gauge stainless-steel cannula was placed ICV according to predetermined coordinates (anterior-posterior, 15.0 mm to the bregma; lateral, 5 mm; horizontal, 214.0 mm to the dura mater). Two stainless-steel screws and cranioplastic cement (Plastics One, Roanoke, VA) secured the cannula. A back-flow of cerebrospinal fluid indicated the cannula was ICV. To facilitate ICV injections, a catheter (Tygon microbore tubing; id, 0.51 mm; od, 1.53 mm; Fisher Scientific) was extended sc from the ICV cannula to the dorsal neck region and exteriorized.

Recombinant porcine TNF-a Porcine TNF-a was cloned into the bacterial expression vector, pGEX-2T (Pharmacia Biotech, Piscataway, NJ). Fusion protein, consisting of cytokine and glutathione-S-transferase, was expressed in Escherichia coli DH5a. A 500-ml culture of recombinant bacteria, amplified from a single colony, was grown in Superbroth with 100 mg/ml ampicillin to an OD600 of approximately 0.7 at 37 C in a 2-liter Fernbach flask. Fusion protein expression was induced with 0.2 mm IPTG. After 4 h at 30 C, cells were harvested by centrifugation at 400 3 g for 15 min at 4 C and resuspended in 25 mm Tris HCl, pH 8.0, 5 mm EDTA, 50 mg/ml phenylmethylsulfonyl fluoride, and 25 mg/ml aprotinin, and frozen at 280 C. The insoluble fusion protein aggregates were isolated by repeated rounds of sonic disruption and centrifugation in the presence of 0.25% Triton X-100 and 0.25% Na deoxycholate in 25 mm Tris HCl, pH 8.0, 5 mm EDTA. Aggregates were solubilized and refolded in 8 m urea, 2 mm arginine, 5 mm EDTA, 0.005% Tween-20, 2 mm reduced glutathione, and 0.02 mm oxidized glutathione. Refolded protein was dialyzed and concentrated on a 30-kDa mw-cutoff membrane (S11Y30, Amicon, Beverly, MA) and passed over a glutathione-sepharose 4B column (Pharmacia Biotech.) in 50 mm Tris HCl, 150 mm NaCl, pH 8.0 (Tris-saline). TNF-a was cleaved on the column with 0.5 ml/ml thrombin (Boehringer Mannheim, Indianapolis, IN) in Tris-saline with 2.5 mm CaCl2 and eluted with Tris-saline. Purity was assessed by Coomassie blue stained SDS-polyacrylamide gels and activity by L929 bioassay (29). Porcine TNF-a was stored frozen (280 C) and later diluted in sterile PBS for ICV injection.

Systemic responses Body temperature (TB). In some pigs after jugular catheterization, temperature-sensitive radio transmitters (Model VHF-T-1, Mini-mitter Co., Inc., Sunriver, OR) were implanted adjacent to a jugular vein. TB was recorded at 20-min intervals using an automated temperature-sensitive radio telemetry system (DATACOL Data Acquisition System Version 5.0, Mini-Mitter Co., Inc.), as previously described (30). Behavioral paradigm. A food-motivated test was used to assess the behavioral effects of LPS ip or TNF-a ICV. A similar test was used previously and was sensitive to LPS in both a time- and dose-dependent fashion (30). In brief, motivation for food was established by removing the food 12 h before administering treatments. Food was returned for a 10-min test period, and behavior was directly monitored by scan sampling at 30-sec intervals for the entire period. Food intake and time spent standing, eating, and somnolent (i.e. time spent in the drowsy state, as determined by recumbency time with both eyes closed) was recorded. Plasma TNF-a and cortisol. Serial blood samples were collected into heparinized syringes (Sarstedt, Inc., Newton, NC) via an indwelling jugular catheter and placed immediately on ice. Whole blood samples were centrifuged (3500 3 g for 15 min at 4 C), and resultant plasma was stored at 280 C until assayed. Total plasma TNF-a was measured using a commercially available enzyme-linked immunosorbent assay specific for porcine TNF-a (En-


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dogen, Inc., Cambridge, MA). Plasma samples were assayed in triplicate at either a 1:1 or 1:10 dilution. The assay has a lower level of sensitivity of 10 pg/ml TNF-a, and the intra- and interassay coefficients of variation were less than 10% and less than 15%, respectively. Total plasma cortisol was measured using a commercially available 125 I RIA kit (ICN Biomedicals, Inc., Costa Mesa, CA). Pooled plasma samples from pigs with high (.100 ng/ml) and low (,25 ng/ml) cortisol were used for validation. Recovery was validated using 20 ml of porcine plasma spiked with 0, 10, 100, or 1000 ng/ml of cortisol standards in a 25-ml reaction vol. Recovery levels ranged from 82–100%. Plasma samples also were serially diluted 1:2, 1:4, and 1:8 with the provided diluent to demonstrate parallelism with mean cortisol levels of 96.9, 81.6, and 71.4 ng/ml, respectively, after adjusting for dilution. Intra- and interassay variations were 7.2% and 9.3%, respectively. Sensitivity of the assay was 1.5 ng/ml.

Experimental protocols Acute responses to peripheral LPS. Twenty pigs, surgically prepared with jugular catheters and temperature-sensitive radio telemeters, were used in a completely randomized design to determine the dose-effect relationship for LPS ip on food intake, behavior, TB, plasma cortisol, and plasma TNF-a. Pigs were fasted 12 h before treatments to enhance motivation for food. LPS from Escherichia coli serotype K-235 (phenol extracted), which we have used in pigs to induce sickness (30, 31), was purchased from Sigma Chemical Co. (St. Louis, MO) and dissolved in sterile pyrogen-free 0.9% NaCl for ip injections. Saline or 0.5, 5.0, and 50 mg/kg LPS was injected ip in a total vol of 2 ml at 0900 h (n 5 5). Serial blood samples were collected via indwelling jugular catheters for plasma cortisol and TNF-a determination. Pigs were subjected to the behavioral test immediately before treatments were administered and again at 2, 4, 8, 12, and 24 h post injection, and TB was monitored at 20-min intervals throughout the experiment. Acute responses to central TNF-a. Six pigs were surgically prepared with iv and ICV cannulas to determine the central effects of TNF-a on food intake, behavior, and plasma cortisol. Pigs were fasted 12 h before treatments to enhance motivation for food. At 0900 h, they were subjected to the food-motivated test to determine the baseline level of food intake and behavioral activity. Immediately after the test, pigs were injected ICV with 100 ml PBS or the same vol PBS containing 5 or 50 ng/kg recombinant porcine TNF-a. Pigs were subjected to the behavioral test again at 2, 4, 8, 12, and 24 h post injection. Serial blood samples were collected via indwelling jugular catheters for determination of plasma cortisol concentration. Each pig was assigned to each of the three treatments (0, 5, or 50 ng/kg TNF-a) after a Latin-square design, with pig and day serving as blocking factors.

Statistical analysis All data analyses were conducted using General Linear Model procedures (32). Data were subjected to ANOVA to determine the significance of main factors and main factor interactions. Cortisol and temperature data were subjected to a two-factor repeated-measures ANOVA. When ANOVA revealed a significant effect of dose or a dose 3 time interaction, differences between treatment means were tested using paired t tests. All data are presented as means 6 se.

Results Peripheral effects of LPS in swine

Peripheral administration of LPS caused a substantial reduction in food intake and an increase in somnolence. Twoway ANOVA of food intake and somnolence revealed a main effect of dose (P , 0.05), time (P , 0.05), and the time 3 dose interaction (P , 0.01). LPS induced marked anorexia, with even the smallest dose reducing, altogether, the motivation of pigs to eat (Fig. 1). Whereas saline-injected pigs engaged in feeding behavior and consumed 175 g of food during the 10-min food-motivated test at 2 h, pigs receiving LPS (0.5, 5, or 50 mg/kg) did not eat. This same feeding pattern was

FIG. 1. Food intake and somnolence of LPS-treated pigs during the 10-min food-motivated tests. Pigs were injected ip with 0, 0.5, 5.0, or 50 mg/kg BW LPS after the food-motivated test at time zero (n 5 5). Values are the mean 6 SE. Asterisks indicate that food intake and somnolence were significantly different, compared with saline controls after LPS (P , 0.05).

evident at 4 h; but at 8 h, pigs that received 0.5 and 5 mg/kg LPS consumed an amount of food similar to the saline control. However, food intake for pigs injected ip with 50 mg/kg did not return to control levels until 24 h (data not shown). In addition to anorexia, hypersomnia was induced by LPS (Fig. 1). During the 10-min test conducted 2 h post injection, pigs receiving saline, 0.5, 5, or 50 mg/kg LPS assumed a somnolent state for 0, 7, 8.5, and 8.75 min, respectively. Somnolent behavior still was evident in LPS-injected pigs at 4 h; but by 8 h, only those receiving the highest dose (50 mg/kg) showed hypersomnia. These changes in behavior caused by LPS were accompanied by an increase in HPA activity as indicated by increased plasma cortisol (Fig. 2). Two-way ANOVA of plasma cortisol concentration revealed a significant effect of time (P , 0.001), dose (P , 0.01), and the time 3 dose interaction (P , 0.01). Before injection, plasma cortisol levels were 25–35 ng/ml, which is consistent with nonstress concentrations for pigs (28). The cortisol response to 0.5 mg/kg LPS, though significantly different from saline control, was small and transient compared with the response to the 5 and 50 mg/kg doses (Fig. 2). In pigs receiving 0.5 mg/kg LPS, cortisol levels peaked at 1.5 h post injection, remained elevated at 2 and 3 h, but returned to baseline by 4 h. In contrast, the cortisol response to 5 and 50 mg/kg LPS was more abrupt and sustained. For example, pigs receiving 50 mg/kg LPS had increased cortisol within 1 h (P , 0.05). Cortisol peaked at 3 h (199 ng/ml) and remained high throughout the sampling period. As anticipated, 5 mg/kg LPS induced an intermediate response.

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The difficulties of combining measures of feeding behavior and TB not withstanding, repeated measures AVOVA of TB revealed a significant time 3 dose interaction (P , 0.05). After each dose of LPS, TB peaked at 4 h (Fig. 3). However, the elevation in TB was not dose-dependent. For example, at 4 h, the change in TB for pigs injected with saline, 0.5, 5, and 50 mg/kg LPS was 0.229, 0.746, 1.283, and 0.836 C, respectively. However, TB for pigs receiving 50 mg/kg LPS was elevated longer. The presentation of food and heat increments of feeding had obvious effects on TB. For instance, pigs that received saline, consumed food when it was offered (Fig. 1), and their TB was increased above baseline after about 2.5 h; pigs that received 5 mg/kg LPS did not eat (Fig. 1) and their TB peaked at 4 h and returned to baseline by 8 h. Injection ip of the high doses of LPS increased plasma TNF-a (Fig. 4). Two-way ANOVA of plasma TNF-a concentration revealed a significant effect of time (P , 0.001), dose (P , 0.01), and the time 3 dose interaction (P , 0.01). Con-

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FIG. 4. Plasma TNF-a concentration after ip LPS. Pigs were injected ip with 0, 0.5, 5.0, or 50 mg/kg BW LPS at time zero (n 5 5). Serial blood samples were collected via an indwelling jugular catheter. Values are the mean 6 SE. Asterisks indicate that plasma TNF-a concentrations were significantly increased after treatment with LPS, compared with saline controls (P , 0.05). Plasma TNF-a for pigs receiving 0.5 mg/kg LPS was undetectable and, therefore, is not shown for clarity of presentation.

sistent with a previous report (31), before injection and after injection of saline or 0.5 mg/kg LPS (data not shown), TNF-a was undetectable by this assay, which has a lower level of detection limit of 10 pg/ml. The TNF-a response to 5 mg/kg LPS was small and transient but was increased at 1.5 h compared with saline (Fig. 4). The TNF-a response to 50 mg/kg LPS was more abrupt and sustained. Pigs receiving 50 mg/kg LPS had increased TNF-a at 1 h (P , 0.01); TNF-a peaked at 1.5 h and was still elevated at 2 h. CNS effects of TNF-a in swine

FIG. 2. Plasma cortisol concentration after ip LPS. Pigs were injected ip with 0, 0.5, 5.0, or 50 mg/kg BW LPS at time zero (n 5 5). Serial blood samples were collected via an indwelling jugular catheter. Values are the mean 6 SE. Asterisks indicate that plasma cortisol concentrations were significantly increased after treatment with LPS, compared with saline controls (P , 0.05).

FIG. 3. Change in TB after ip LPS. Pigs were injected ip with 0, 0.5, 5, or 50 mg/kg BW LPS at time zero. TB was measured at 20-min intervals using an automated radio telemetry system. Pigs had ad libitum access to food for 10-min periods at 0, 2, 4, and 8 h post injection. Data are presented as the change in TB 6 SE from time zero. Asterisks indicate that TB was significantly increased after treatment with LPS, compared with saline controls (P , 0.05).

Two-way ANOVA of food intake and somnolence revealed a main effect of dose (P , 0.05), time (P , 0.001), and time 3 dose interaction (P , 0.01). Central injection of TNF-a (5 and 50 ng/kg) markedly reduced food intake and increased somnolence at 2 and 4 h post injection (Fig. 5). Time spent eating was proportional to food intake, and therefore, data are not shown. To ensure that these effects were caused by TNF-a and not by contaminating endotoxin, the cytokine preparation was heated to 90 C for 15 min. This treatment denatures protein but has no effect on the biological activity of endotoxin. Three separate pigs were injected ICV with 100 ng/kg heat-inactivated TNF-a, an amount 2-fold greater than the highest dose of biologically active cytokine employed in the present study. The injectate containing heatdenatured TNF-a had no effect on either food intake or somnolence (data no shown), indicating that endotoxin contamination was minimal and not responsible for the change in behavior observed after central administration of intact protein. The overt response to central TNF-a was accompanied by increased activity of the HPA axis, as indicated by increased plasma concentration of cortisol (Fig. 6). Twoway ANOVA of plasma cortisol concentration revealed a main effect of dose (P , 0.001), time (P , 0.01), and a time 3 dose interaction (P , 0.01). Cortisol levels for pigs receiving PBS ICV did not rise above baseline, indicating that the injection procedures did not induce stress.


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Discussion

FIG. 5. Effects of centrally administered recombinant porcine TNF-a on food intake and somnolence in pigs during the 10-min food-motivated tests. Pigs were injected ICV with 0, 5, or 50 ng/kg BW TNF-a after the food-motivated test at time zero (n 5 6). Values are the mean 6 SE. Asterisks indicate that food intake and somnolence after TNFa were different, compared with saline controls (P , 0.05).

FIG. 6. Effects of centrally administered TNF-a on plasma cortisol concentration. Pigs were injected ICV with 0, 5, and 50 ng/kg BW TNF-a at time zero. Serial blood samples were collected via an indwelling jugular catheter. Values are the mean 6 SE. Asterisks indicate that plasma cortisol concentration was different after treatment with 50 ng/kg TNF-a, compared with saline controls (P , 0.05).

Whereas 5 ng/kg TNF-a induced anorexia and somnolence (Fig. 5), this dose did not increase cortisol secretion (Fig. 6), suggesting that central TNF-a is more effective in inducing sickness than stimulating the neuroendocrine system. The 50 ng/kg dose of TNF-a, however, induced an abrupt increase in plasma cortisol. In pigs receiving 50 ng/kg TNF-a, plasma cortisol peaked at 1.5 h and remained elevated for 4 h.

This study confirms that pigs respond to ip LPS by reducing food intake, becoming febrile and somnolent, and with an increase in the secretion of cortisol. It also shows that LPS increases plasma levels of TNF-a in swine and confirms that this cytokine, when injected directly into the CNS, is sufficient to induce anorexia, somnolence, and secretion of cortisol. The important findings were that: 1) a low dose of LPS administered ip, which induced marked sickness behavior and fever, failed to increase plasma TNF-a and induced only a small transient increase in plasma cortisol; and 2) a low dose of TNF-a given ICV, which induced marked sickness behavior, did not increase plasma cortisol. These results therefore suggest not only a dissociation between the secretion of TNF-a and the induction of sickness but also between the behavioral and neuroendocrine effects of TNF-a in the brain. The change in behavior and neuroendocrine secretions associated with sickness are well-recognized components of the acute-phase response (5, 33). They are nonspecific to the invading pathogen and are attributed to cytokines released by activated macrophages. The HPA axis in the rat is one of the first systems to respond to LPS. The increase in plasma glucocorticoids is part of an important inhibitory mechanism that modulates immunological and inflammatory responses (34, 35). Indeed, it was recently reported that a low dose of LPS administered ip in rats maximally stimulated the HPA axis but failed to depress social behavior (24), a form of motivation used to quantify sickness (36). It was postulated, and later demonstrated, that the marked increase in plasma corticosterone in response to the low dose of LPS was responsible for precluding sickness behavior. Therefore, at least in the rat, the sensitivity of the HPA axis to inflammatory stimuli is important to prevent behavioral disturbances in response to low-grade immune stimulation. An important attribute of the pig is that behavior, immune, and neuroendocrine systems can be evaluated simultaneously and chronologically in an integrated whole-animal model (28). In the present study, the model afforded us the opportunity to characterize the coincidental changes in behavior, plasma cortisol, and plasma TNF-a after peripheral injection of LPS. Pigs were injected ip with four doses of LPS (i.e. 0, 0.5, 5, and 50 mg/kg) to develop complete dose-response curves for several behavioral and physiological effects (i.e. food intake, somnolence, fever, and cortisol secretion). These doses are substantially less than those typically used in rats and mice because, like human and nonhuman primates, pigs are extremely sensitive to LPS (37). The same doses and serotype of LPS previously were used in pigs to show that the induction of sickness behavior by LPS involved a PG-dependent mechanism (30). Consistent with that report, in the present study, pigs responded to ip LPS with behavioral and physiological responses indicative of an acute gram-negative bacterial infection. After ip administration of even the smallest dose of LPS (0.5 mg/kg), pigs were anorectic, somnolent, and febrile (Figs. 1 & 3). These responses were evident even after 4 h. The increase in plasma cortisol after the same low dose of LPS was less dramatic (Fig. 2). Therefore, contrary to our previous study in rats (which

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showed a marked increase in plasma corticosteroids but no overt symptoms of sickness), after an ip injection of LPS, the pig showed marked sickness behavior but only a small transient increase in cortisol. The small but significant increase in cortisol may have had a permissive effect on behavioral targets (i.e. the CNS), therefore contributing to the marked behavioral response to LPS. However, several studies indicate that adrenalectomized animals are more sensitive to the behavioral effects of LPS or IL-1b than intact controls (24 –26, 38). Alternatively, because corticosteroids have been found to inhibit several effects of LPS, including fever (39, 40) and sickness behavior (24 –26, 38), the relative insensitivity of the pig’s HPA axis to LPS may partially explain its noted sensitivity to the behavioral effects of LPS. In the present study, we also sought to measure TNF-a in plasma of pigs after ip injection of LPS. Although the increase in TNF after injection of LPS is well described for mice and rats, to our best knowledge, only two studies have reported plasma concentrations of TNF in swine. Recently, we reported that pigs injected ip with LPS (5 mg/kg) had increased plasma concentrations of TNF-a, IL-6, and cortisol (31). Because the objective of that study was to determine the relationship between plasma cytokines and alterations in macronutrient metabolism, pigs were fasted, and behavior was not monitored. In another study (41), pigs were anesthetized, and 5 mg/kg BW LPS was continuously infused into the superior mesenteric artery over a 60-min period. Again, TNF increased after intraarterial infusion of LPS, and high plasma TNF was positively correlated with lethality caused by endotoxic shock (41). In the present study, pigs were prepared with indwelling jugular catheters, so that serial blood samples could be obtained while simultaneously monitoring behavior. In addition, pigs were implanted with radio telemeters for periodic undisturbed monitoring of TB. The 50-mg/kg dose of LPS induced a rapid increase in plasma TNF-a, with levels being significantly elevated at 1 h, peaking at 1.5 h, and still high (compared with saline control) at 2 h (Fig. 4). Comparatively, 5 mg/kg induced a moderate increase in TNF-a, which was significant at 1.5 h only. Despite inducing fever and sickness behavior for more than 4 h, the 0.5 mg/kg dose did not increase plasma TNF-a. This is consistent with an earlier report, where the same dose of the LPS also failed to increase plasma concentration of TNF-a (31). There is, therefore, a dissociation between the induction of sickness behavior by LPS and high circulating levels of TNF-a. It is of note that regardless of a purported link between TNF-a and cachexia, there is no clear correlation between weight loss and plasma TNF-a in cachectic patients (42). Of course, that TNF-a was not elevated in response to 0.5 mg/kg LPS does not preclude the possibility that other cytokines, such as IL-1 or IL-6, were. However, in a previous study, the same dose of LPS given ip to pigs also failed to increase plasma IL-6 (31). A more plausible explanation for the low dose of LPS inducing sickness without increasing plasma TNF-a is that cytokines produced in the periphery act locally on sensory neurons, which when activated, induce cytokines to be produced centrally. In support of this hypothesis, ip injection of LPS increased messenger RNAs (mRNAs) encoding TNF-a, IL-1, and IL-6 in murine brain (10). Furthermore, subdiaphragmatic vagotomy prevented the in-

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creased expression of cytokine mRNAs in mouse brain and blocked the normal depression in social behavior (43) and anorexia (44) caused by LPS. Of particular note is the finding that although vagotomy blocked LPS-induced sickness behavior, it did not prevent the increase in plasma IL-1 (43). Thus, increased cytokines in plasma do not ensure sickness, nor does their absence preclude it. The behavioral response seems dependent upon cytokines in the CNS. To confirm this in pigs (and in doing so, take advantage of the pig model to simultaneously and chronologically evaluate behavioral and physiological responses to a cytokine administered directly into the CNS), recombinant porcine TNF-a was injected ICV into unrestrained pigs. TNF-a has not been measured in CSF of pigs, but the doses injected ICV (i.e. 5 and 50 ng/kg) were projected to be equal to or lower than that needed to achieve pathophysiologic concentrations in the cerebrospinal fluid of animal models of disease or humans with viral or bacterial meningitis (45). The doses also were sufficiently low to ensure that centrally injected cytokine would not diffuse into peripheral blood, although plasma was not assayed for TNF-a after ICV injection. The present results support the idea that many neural effects of inflammatory challenge are mediated by cytokines, like TNF-a, acting directly in the CNS. When administered into the CNS, porcine TNF-a, at a dose as low as 5 ng/kg, induced a transient but marked reduction in food intake and an increase in somnolence. The anorectic properties of this cytokine are apparent, because a reduction in food intake was evident, even though pigs were subjected to a 12-h food withdrawal period before ICV injection. The use of homologous cytokine was important because, e.g. in mice and rats, recombinant human TNF-a binds only the p55 receptor and not the p75 receptor. This may explain why, in previous studies, the rat was relatively insensitive to TNF-a (i.e. human) injected ICV (13, 45). Consistent with our finding that the pig is more sensitive to the behavioral effects of LPS than to the neuroendocrine effects of LPS, 5 ng/kg TNF-a injected ICV reduced food intake and behavioral activity without increasing plasma cortisol concentration. Again, if the HPA axis is not as responsive to TNF-a as our results suggest, this may account for the profound behavioral effects of central TNF-a. In summary, our results confirm that, in the pig, LPS induces the secretion of TNF-a and that this cytokine can act directly in the brain to elicit behavioral and physiological effects associated with inflammatory challenge. They also suggest a dissociation between the behavioral and neuroendocrine effects of inflammatory stimuli in the periphery and CNS. These data therefore indicate that the different systems, which are responsive to inflammatory stimuli, exhibit different sensitivities. It is suggested that the relative insensitivity of the pig’s HPA axis to LPS and TNF-a is, at least partially, responsible for the marked behavioral effects that were observed. References 1. McGeehan GM, Becherer JD, Bast RC, Boyer CM, Champion B, Conolly KM, Conway JG, Furdon P, Karp S, Kidao S, McElroy AB, Nichols J, Pryzwansky KM, Schoenen F, Sekut L, Truesdale A, Verghese M, Warner J, Ways JP 1994 Regulation of tumor necrosis factor-a processing by a metalloproteinase inhibitor. Nature 370:558 –561

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